U.S. patent application number 17/086817 was filed with the patent office on 2021-02-18 for method for improved diagnosis of dysplasias.
The applicant listed for this patent is Ventana Medical Systems, Inc.. Invention is credited to Richard Batrla, Anja Reichert, Ruediger Ridder, Marcus Trunk-Gehmacher.
Application Number | 20210047697 17/086817 |
Document ID | / |
Family ID | 1000005190925 |
Filed Date | 2021-02-18 |
United States Patent
Application |
20210047697 |
Kind Code |
A1 |
Ridder; Ruediger ; et
al. |
February 18, 2021 |
METHOD FOR IMPROVED DIAGNOSIS OF DYSPLASIAS
Abstract
The present invention relates to a method for improved diagnosis
of dysplasias based on simultaneous detection of INK4a gene
products and at least one marker for cell proliferation.
Particularly the present invention provides a method for
discriminating dysplastic cells over-expressing INK4a gene products
from cells over-expressing INK4a gene products without being
dysplastic by detection of a marker suitable for characterizing the
proliferation properties of the respective cell. The
characterization of the proliferation properties may comprise the
detection of a marker or a set of markers characteristic for active
cell proliferation and/or a marker or a set of markers
characteristic for retarded or ceased cell proliferation. The
method presented herein thus enables for a specific diagnosis of
dysplasias in histological and cytological specimens.
Inventors: |
Ridder; Ruediger;
(Schriesheim, DE) ; Reichert; Anja; (Nussloch,
DE) ; Trunk-Gehmacher; Marcus; (Heidelberg, DE)
; Batrla; Richard; (Allschwil, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ventana Medical Systems, Inc. |
Tucson |
AZ |
US |
|
|
Family ID: |
1000005190925 |
Appl. No.: |
17/086817 |
Filed: |
November 2, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16579495 |
Sep 23, 2019 |
|
|
|
17086817 |
|
|
|
|
13799093 |
Mar 13, 2013 |
|
|
|
16579495 |
|
|
|
|
12289487 |
Oct 29, 2008 |
8975036 |
|
|
13799093 |
|
|
|
|
10533384 |
Jun 8, 2006 |
|
|
|
PCT/EP03/50738 |
Oct 21, 2003 |
|
|
|
12289487 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/57411 20130101;
G01N 33/574 20130101; G01N 33/57484 20130101; C12Q 1/6886
20130101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; G01N 33/574 20060101 G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 28, 2002 |
EP |
02024030.5 |
Mar 7, 2003 |
EP |
03100584.6 |
Claims
1. A kit consisting of (i) a pair of primary antibodies, and (ii)
optionally detection agents for performing an immunoenzymatic
assay, the pair of primary antibodies consisting of an anti-human
p16.sup.INK4a monoclonal antibody and an anti-human Ki67 monoclonal
antibody.
2. The kit of claim 1, wherein the anti-human p16.sup.INK4a
monoclonal antibody is a mouse monoclonal antibody, and wherein the
anti-human Ki67 monoclonal antibody is a rabbit monoclonal
antibody.
3. The kit of claim 1, wherein the anti-human p16.sup.INK4a
monoclonal antibody and the anti-human Ki-67 monoclonal antibody
are provided as a cocktail.
3. The kit of claim 1, further comprising a secondary antibody
specific to the anti-human p16.sup.INK4a monoclonal antibody,
wherein the secondary antibody specific to the anti-human
p16.sup.INK4a monoclonal antibody is conjugated to a peroxidase
enzyme.
4. The kit of claim 1, further comprising a secondary antibody
specific to the anti-human Ki-67 monoclonal antibody, wherein the
secondary antibody specific to the anti-human Ki-67 monoclonal
antibody is conjugated to an alkaline phosphatase enzyme.
5. The kit of claim 1, further comprising a first chromogenic agent
and a second chromogenic agent.
6. The kit of claim 5, wherein the first chromogenic agent produces
brown signals; and wherein the second chromogenic agent produces
red signals.
7. The kit of claim 5, wherein the first chromogenic agent is
3,3'-diaminobenzidine, and wherein the second chromogenic agent is
FastRed.
8. The kit of claim 1, wherein the kit consists of the pair of
primary antibodies.
9. The kit of claim 1, wherein the anti-human p16.sup.INK4a
monoclonal antibody is specific to a human p16.sup.INK4a protein in
a human cervical specimen; and wherein the anti-human Ki-67
monoclonal antibody is specific to a human Ki-67 protein in the
human cervical specimen.
10. The kit of claim 9, wherein the human cervical specimen is a
cytological specimen derived from the cervix uteri.
11. A kit comprising (i) a pair of primary antibodies, the pair of
primary antibodies consisting of an anti-human p16.sup.INK4a
monoclonal antibody and an anti-human Ki67 monoclonal antibody;
(ii) 3,3'-diaminobenzidine; and (iii) a chromogenic agent which
produces red signals.
12. The kit of claim 11, wherein the chromogenic agent which
produces the red signals is FastRed.
13. The kit of claim 11, wherein the anti-human p16.sup.INK4a
monoclonal antibody is a mouse monoclonal antibody, and wherein the
anti-human Ki67 monoclonal antibody is a rabbit monoclonal
antibody.
14. The kit of claim 11, wherein the anti-human p16.sup.INK4a
monoclonal antibody is specific to a human p16.sup.INK4a protein in
a human cervical specimen; and wherein the anti-human Ki-67
monoclonal antibody is specific to a human Ki-67 protein in the
human cervical specimen.
15. The kit of claim 14, wherein the human cervical specimen is a
cytological specimen derived from the cervix uteri.
16. The kit of claim 11, further comprising a secondary antibody
specific to the anti-human p16.sup.INK4a monoclonal antibody; and a
secondary antibody specific to the anti-human Ki-67 monoclonal
antibody.
17. The kit of claim 16, wherein the secondary antibody specific to
the anti-human p16.sup.INK4a monoclonal antibody is associated with
a peroxidase enzyme; and wherein the secondary antibody specific to
the anti-human Ki-67 monoclonal antibody is associated with an
alkaline phosphatase enzyme.
18. The kit of claim 16, wherein the secondary antibody specific to
the anti-human p16.sup.INK4a monoclonal antibody is conjugated to a
peroxidase enzyme; and wherein the secondary antibody specific to
the anti-human Ki-67 monoclonal antibody is conjugated to an
alkaline phosphatase enzyme.
19. The kit of claim 11, wherein the kit consists of (i) the pair
of primary antibodies, (ii) the 3,3'-diaminobenzidine; and (iii)
the chromogenic agent which produces red signals.
20. A kit comprising: a pair of primary antibodies, the pair of
primary antibodies consisting of an anti-human p16.sup.INK4a
monoclonal antibody and an anti-human Ki-67 monoclonal antibody; a
secondary antibody specific to the anti-human p16.sup.INK4a
monoclonal antibody, wherein the secondary antibody specific to the
anti-human p16.sup.INK4a monoclonal antibody is associated with a
peroxidase enzyme; and a secondary antibody specific to the
anti-human Ki-67 monoclonal antibody, wherein the secondary
antibody specific to the anti-human Ki-67 monoclonal antibody is
associated with an alkaline phosphatase enzyme.
21. The kit of claim 20, wherein the secondary antibody specific to
the anti-human p16.sup.INK4a monoclonal antibody is conjugated to a
peroxidase enzyme; and wherein the secondary antibody specific to
the anti-human Ki-67 monoclonal antibody is conjugated to an
alkaline phosphatase enzyme.
22. The kit of claim 20, wherein the anti-human p16.sup.INK4a
monoclonal antibody is a mouse monoclonal antibody, and wherein the
anti-human Ki-67 monoclonal antibody is a rabbit monoclonal
antibody.
23. The kit of claim 20, further comprising a first chromogenic
agent and a second chromogenic agent.
24. The kit of claim 23, wherein the first chromogenic agent is
3,3'-diaminobenzidine.
25. The kit of claim 23, wherein the second chromogenic agent is
FastRed.
26. The kit of claim 20, wherein the anti-human p16.sup.INK4a
monoclonal antibody is specific to a human p16.sup.INK4a protein in
a human cytological specimen; and wherein the anti-human Ki-67
monoclonal antibody is specific to a human Ki-67 protein in the
human cytological specimen.
27. The kit of claim 20, wherein the kit consists of the pair of
primary antibodies, the secondary antibody specific to the
anti-human p16.sup.INK4a monoclonal antibody, and the secondary
antibody specific to the anti-human Ki-67 monoclonal antibody.
28. A kit comprising: an antibody cocktail consisting of an
anti-human p16.sup.INK4a monoclonal antibody and an anti-human
Ki-67 monoclonal antibody; a first chromogen that produces brown
signals; and a second chromogen that produces red signals.
29. The kit of claim 28, wherein the anti-human p16.sup.INK4a
monoclonal antibody is a mouse monoclonal antibody, and wherein the
anti-human Ki-67 monoclonal antibody is a rabbit monoclonal
antibody.
30. The kit of claim 28, wherein the anti-human p16.sup.INK4a
monoclonal antibody is specific to a human p p16.sup.INK4a protein
in a human cervical specimen; and wherein the anti-human Ki-67
monoclonal antibody is specific to a human Ki-67 protein in the
human cervical specimen; and wherein the human cervical specimen is
a cytological specimen derived from the cervix uteri.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 16/579,495 filed on Sep. 23, 2019, which application is a
continuation of U.S. Ser. No. 13/799,093, filed Mar. 13, 2013,
which is a divisional application of U.S. Ser. No. 12/289,487,
filed Oct. 29, 2008, now U.S. Pat. No. 8,975,036, which application
is a divisional application of U.S. Ser. No. 10/533,384, now
abandoned, which is a National Stage of International Application
PCT/EP03/050738, filed Oct. 21, 2003, published May 6, 2004, under
PCT Article 21(2) in English; which claims the priority of EP
02024030.5, filed Oct. 28, 2002; and EP 03100584.6, filed Mar. 7,
2003, the disclosures of which are each hereby incorporated by
reference herein in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for improved
diagnosis of dysplasias based on simultaneous detection of INK4a
gene products and at least one marker for cell proliferation. In
particular, the present invention provides a method for
discriminating dysplastic cells over-expressing INK4a gene products
from cells over-expressing INK4a gene products without being
dysplastic by detection of a marker suitable for characterising the
proliferation properties of the respective cell. The
characterisation of the proliferation properties may comprise the
detection of a marker or a set of markers characteristic for active
cell proliferation and/or a marker or a set of markers
characteristic for retarded or ceased cell proliferation. The
method presented herein thus enables a specific diagnosis of
dysplasias in histological and cytological specimens.
[0003] The detection of the over-expression of p16.sup.INK4a in
biological samples has proven as a useful marker in the detection
of anogenital lesions, such as carcinoma of the uterine cervix (see
WO00/01845; Klaes et al., Int. J. Cancer: 92, 276-284 (2001)). The
method based on p16.sup.INK4a-specific immuno-chemical staining
allows for a sensitive and specific identification of dysplastic
cells in tissue sections and in cytological samples.
[0004] In immuno-histochemical examinations of tissues, dysplastic
and neoplastic cells can be stained using a p16.sup.INK4a specific
antibody mediated staining procedure. The histological diagnosis of
neoplastic lesions can thus be supported by a staining procedure
based on a molecular marker characteristic for transformation of
cells in anogenital lesions. The diagnosis, whether or not cells
are neoplastic, in these procedures is not solely based on the
p16.sup.INK4a specific staining, but does also rely on the
histological information.
[0005] This is due to the fact that, in about 20-30% of samples,
metaplastic cells show some immunoreactivity with p16.sup.INK4a
specific antibodies, and thus are stained in the course of the
procedures. Yet the staining pattern of these metaplastic cells
differs from the pattern of neoplastic lesions. Metaplastic cells
give rise to a patchy or focal staining pattern, whereas neoplastic
lesions give rise to diffuse staining pattern. Moreover, the
staining intensities of metaplastic cells are predominantly less
than that of neoplastic cells.
[0006] The common methods used in screening tests for the early
detection of dysplasias and/or neoplasias do not employ histology
based tests, but rather rely on cytological testing procedures. Yet
especially in cases when there is no histological information
available concerning the architecture of tissues, such as, for
example, in cytological examinations, testing for p16.sup.INK4a
over-expression alone may lead to false positive results. This is
due to the fact that those fractions of metaplastic cells
expressing p16.sup.INK4a at detectably elevated levels may not be
differentiated by means of a histologic criteria.
[0007] The percentage of cells showing over-expression of
p16.sup.INK4a increases in the course of emergence of dysplasias.
So, in neoplastic or pre-neoplastic stages, when only a restricted
population of neoplastic or pre-neoplastic cells is present in
samples, the immunoreactivity of p16.sup.INK4a may be weak. This
weak immunoreactivity may be of about the level as the level caused
by metaplastic cells. In later stages of dysplasias, the overall
immunoreactivity of p16.sup.INK4a is stronger, so neoplastic
lesions are easily discernible from metaplasias even in a
cytological testing format. This might lead to cases where the
presence of metaplastic cells expressing p16.sup.INK4a might be
confused with the presence of neoplastic cells, and thus produce a
false positive result.
[0008] Especially in screening tests, where the detection of early
stages of neoplasias is desirable, this condition is quite
unpleasant. This is especially true, as the p16.sup.INK4a based
diagnosis has proven to be a valuable tool in histological
examinations and the application in cytological based screening
procedures would be able to enhance these established
procedures.
[0009] To reduce false positive results in cytological testing
formats and thereby further enhance the fidelity of the
p16.sup.INK4a mediated diagnosis of anogenital lesions, a method
for discriminating the metaplasias from neoplastic and dysplastic
lesions would be desirable. A method for the discrimination of
metaplasias from neoplastic and pre-neoplastic lesions is provided
within the embodiments claimed according to the present
invention.
[0010] For supporting the discrimination of metaplasias from
neoplastic lesions in testing procedures based on the
over-expression of p16.sup.INK4a, a marker molecule would be
desirable that is expressed in neoplastic and/or pre-neoplastic
cells and tissues and which is not expressed simultaneously with
INK4a gene products in one single metaplastic cell.
[0011] A solution to the problem present in the art is provided by
the methods claimed according to this invention. In the course of
the experiments leading to the present invention, the inventors
have found that a combination of detection of the presence or
absence and/or the level of p16.sup.INK4a, in combination with at
least one marker for cell proliferation, such as e.g. Ki67, Ki-S2,
mcm5 or mcm2, may solve the problem present in the art.
[0012] In the art, a couple of documents pertaining to the use of a
combination of molecular markers for improved diagnosis of
dysplasias is presented. In WO0208764, a method for improved
diagnosis of cervical malignancies is disclosed using a combination
of an HPV marker and a marker for cell proliferation or viral
activity. p16.sup.INK4a is mentioned in the context of this
invention as a viral activity marker to be combined with HPV
markers.
[0013] In EP1217377, a method for automated detection of cervical
malignancies is disclosed that is mediated by detection of more
than one marker molecule. Some defined marker combinations are
named within the document. There is no disclosure in this document
relating to the choice of suitable markers for a combination. The
purpose of the combination in this application is improved fidelity
of the automated analysis of staining patterns in biological
cytological specimens. This document mentions combination of
p16.sup.INK4a with other tumor markers.
[0014] WO02059616 discloses a method for detection of cell-cycle
disturbances for improved diagnosis of cervical malignancies. The
document discloses that dysplastic cells exhibit disturbances in
cell cycle control, and may thus be identified by means of
detection of cyclin E type proteins together with post G1
substances in cells.
[0015] Jeffrey Keating in "Ki67, Cyclin E, and p16.sup.INK4a Are
Complimentary Surrogate Biomarkers for human papilloma
Virus-Related Cervical Neoplasia" (American Journal of Surgical
Pathology 25(7): 884-891, (2001)) discloses the complementarity of
the use of p16.sup.INK4a, Ki67, and Cyclin E in the course of
diagnosis of cervical dysplasias. The document refers to the
problems of each single marker in the detection of dysplasias, and
states that p16.sup.INK4a in combination with Cyclin E may be
suitable to overcome the drawbacks of the single marker molecule,
especially when cytological specimens are under examination. No
disclosure teaching use of p16.sup.INK4a in concert with a marker
characteristic of proliferating cells such as Ki67 is given. The
document does not teach the combination of p16.sup.INK4a for use in
diagnostic methods; moreover, the disclosure pertains to a
restricted use of Ki67 in cervical differential diagnosis, and
thus, teaches away from the use of this marker in diagnosis of
cervical malignancies.
[0016] In the art, there is no disclosure teaching the use of a
combination of p16.sup.INK4a with a marker characteristic of cell
proliferation for use in a diagnostic method for improved
discrimination of p16.sup.INK4a positive non-dysplastic cells from
p16.sup.INK4a positive dysplasias. WO02059616 does not give a hint
as to the use of p16.sup.INK4a in the detection of dysplastic cell
proliferation. EP1217377 does not teach a purpose for the
combination of tumor markers in the course of the detection other
than the automation of the analysis process. There is no disclosure
pertaining to the advantage of combinations for specific
discrimination purposes such as discrimination of dysplastic cells
from e.g. metaplastic cells in cervical specimens.
SUMMARY OF THE INVENTION
[0017] The inventors of the present invention sought to overcome
the drawback present in the art that p16.sup.INK4a, over-expressed
in various dysplasias, may also be detected in some other
non-dysplastic cells. The discrimination between non-dysplastic
p16.sup.INK4a positive cells and dysplastic cells over-expressing
p16.sup.INK4a may be based on the proliferation characteristics of
the respective cells. In normal cells, p16.sup.INK4a inhibits cdk4,
and thus inhibits proliferation. In contrast, in dysplastic cells,
this regulation is impaired. Thus, p16.sup.INK4a does not
lead--despite its uncommonly high expression level--to an
inhibition of the cell proliferation.
[0018] The inventors of the present invention found that dysplastic
cells may be discriminated from cells exhibiting controlled cell
proliferation by the simultaneous detection of p16.sup.INK4a with a
marker characteristic for cell proliferation. Due to the fact that,
in normal cells, elevated levels of p16.sup.INK4a inhibit cell
proliferation, cells over-p16.sup.INK4a may be classified as being
dysplastic provided they are exhibiting the characteristics of
active cell proliferation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with drawing(s) will be provided by the Office upon
request and payment of the necessary fee.
[0020] FIG. 1 is an example of fluorescent staining of a
histological specimen of the cervix uteri. FIG. 1 shows staining of
a severe dysplasia using antibodies directed against p16.sup.INK4a.
For experimental details, see Example 1. Immunoreactivity for
p16.sup.INK4a renders green fluorescence. Almost all cells of the
lesion are diffusely positively stained in the cytoplasm and in the
nuclei.
[0021] FIG. 2 is an example of fluorescent staining of a
histological specimen of the cervix uteri. FIG. 2 shows staining of
a severe dysplasia using antibodies directed against Ki67. For
experimental details, see Example 1. Immunoreactivity for Ki67
renders red fluorescence. Many cells of the dysplasia show nuclear
positivity for Ki67.
[0022] FIG. 3 is an example of fluorescent double staining of a
histological specimen of the cervix uteri. FIG. 3 shows staining of
a severe dysplasia using antibodies directed against Ki67 and those
directed against p16.sup.INK4a For experimental details, see
Example 1. Immunoreactivity for Ki67 renders red fluorescence,
immunoreactivity for p16.sup.INK4a renders green fluorescence and
the overlay of red and green fluorescence renders yellow
fluorescence. Many cells of the dysplasia show nuclear positivity
for Ki67, as well as positivity for p16.sup.INK4a, and thus give
rise to yellow fluorescence.
[0023] FIG. 4 is an example of fluorescent staining of a
histological specimen of the cervix uteri. FIG. 4 shows staining of
a squamous metaplasia using antibodies directed against
p16.sup.INK4a. For experimental details, see Example 1.
Immunoreactivity for p16.sup.INK4a renders green fluorescence. Some
cells of the metaplasia stain diffusely positive in the cytoplasm
and in the nuclei.
[0024] FIG. 5 is an example of fluorescent staining of a
histological specimen of the cervix uteri. FIG. 5 shows staining of
a squamous metaplasia using antibodies directed against Ki67. For
experimental details, see Example 1. Immunoreactivity for Ki67
renders red fluorescence. Many cells of the squamous metaplasia
show nuclear positivity for Ki67. Areas that have been identified
as positively expressing p16.sup.INK4a do not show any positive
staining for Ki67, indicating lack of expression of the antigen in
these areas.
[0025] FIG. 6 is an example of fluorescent double staining of a
histological specimen of the cervix uteri. FIG. 6 shows staining of
a squamous metaplasia using antibodies directed against Ki67 and
against p16.sup.INK4a. For experimental details, see Example 1.
Immunoreactivity for Ki67 renders red fluorescence,
immunoreactivity for p16.sup.INK4a renders green fluorescence and
the overlay of red and green fluorescence renders yellow
fluorescence. Areas that are positively expressing p16.sup.INK4a do
not show any positive staining for Ki67, indicating lack of
expression of the antigen in these areas. No cells in the specimen
give rise to yellow fluorescence.
[0026] FIG. 7 is an example of chromogenic double staining of a
histological specimen of the cervix uteri. FIG. 7 shows staining of
a severe dysplasia using antibodies directed against Ki67 and
against p16.sup.INK4a. For experimental details, see Example 6.
Immunoreactivity for Ki67 renders red nuclear staining,
immunoreactivity for p16.sup.INK4a renders brownish staining over
the whole cell, and double staining renders brown cells with red
nuclei. Many cells of the dysplasia show nuclear positivity for
Ki67, as well as positivity for p16.sup.INK4a, and thus, give rise
to a pattern of brown cells with red nuclei.
[0027] FIG. 8 is an example of chromogenic double staining of a
cytological specimen of the cervix uteri. FIG. 8 shows staining of
a severe dysplasia using antibodies directed against Ki67 and
against p16.sup.INK4a. For experimental details, see Example 7.
Immunoreactivity for Ki67 renders red nuclear staining,
immunoreactivity for p16.sup.INK4a renders brownish staining over
the whole cell, and double staining renders brown cells with red
nuclei. Many cells of the dysplasia show nuclear positivity for
Ki67 as well as positivity for p16.sup.INK4a, and thus, give rise
to a pattern of brown cells with red nuclei.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention relates to a method for discrimination
of neoplastic, pre-neoplastic and/or dysplastic lesions from
non-dysplastic cells showing elevated levels of p16.sup.INK4a, in
biological samples in a histological or cytological testing
procedure based on the detection of the presence or absence of
cells expressing p16.sup.INK4a gene simultaneously with cell
proliferation markers in said biological samples. Suitable markers
for cell proliferation may be, e.g., Ki67, Ki-S2, Ki-S5, mcm5, or
mcm2. In one embodiment of the invention, protein or mRNA of the
markers for cell proliferation may serve as a marker for
discrimination of metaplasias from early dysplastic or
pre-neoplastic lesions in samples.
[0029] One aspect of the present invention is to provide a method
for discriminating dysplastic cells over-expressing INK4a gene
products from other cells expressing INK4a gene products at a
detectable level in biological samples comprising determining in a
cytological or histological testing procedure the co-expression of
at least two marker molecules in at least one single cell, wherein
at least one marker molecule is an expression product encoded by
the INK4a gene and at least one further marker molecule is a cell
proliferation marker, wherein the over-expression of at least one
INK4a gene product and expression of at least one marker for active
cell proliferation at an immuno-chemically detectable level within
said single cell is indicative of the dysplastic state of the cell,
and wherein the over-expression of at least one INK4a gene product
and expression of at least one marker for senescence, terminal
differentiation of cells, apoptosis, or cell cycle arrest at a
detectable level within said single cell is indicative of the
non-dysplastic state of the cell.
[0030] A second aspect of the present invention relates to a test
kit for determination of dysplasias in samples according to the
method disclosed herein.
[0031] During the experiments leading to the present invention, it
was found that, under certain circumstances, non-dysplastic cells
may show immunoreactivity for p16.sup.INK4a. The inventors found
that these cells exhibiting ordered control of cell proliferation
in response to the elevated levels of the INK4a gene products are
subject to growth arrest. Thus, these individual cells do not show
immunoreactivity for markers of cell proliferation. In contrast,
transformed cells over-expressing p16.sup.INK4a exhibit
dysregulated control of cell proliferation, and do not respond to
the elevated level of p16.sup.INK4a by cessation of proliferation.
Thus, these dysplastic cell show simultaneous expression of
p16.sup.INK4a with markers for cell proliferation. The inventors
thus found that a simultaneous detection of p16.sup.INK4a with
markers for cell proliferation may serve to discriminate dysplastic
cells from arrested cells over-expressing p16.sup.INK4a such as,
e.g., metaplastic cells.
[0032] The discovery that p16.sup.INK4a over-expressed in various
dysplasias may also be detected in some other non-dysplastic cells
led the inventors of the presented method to establish a technique
for discrimination between non-dysplastic p16.sup.INK4a positive
cells and dysplastic cells over-expressing p16.sup.INK4a based on
the proliferation characteristics of the respective cells. Whereas
in normal cells p16.sup.INK4a inhibits cdk4, and thus inhibits cell
proliferation, this is not true for dysplastic cells. Thus, in
dysplastic cells p16.sup.INK4a does not lead--despite its
uncommonly high expression level--to an inhibition of the cell
proliferation.
[0033] The method disclosed herein is based on the fact that
dysplastic cells may be discriminated from cells exhibiting normal
cell proliferation by the simultaneous detection of an INK4a gene
product such as, e.g., p16.sup.INK4a, with a marker characteristic
for cell proliferation.
[0034] The term marker, as well as marker molecules in general,
shall be used herein to pertain to proliferation marker gene
expression products as well as to INK4a gene expression
products.
[0035] The denominations given throughout this text for genes may,
in part, relate to the genes or proteins as they have been
discovered from any organism. In the context of the present
invention, this denomination shall confer to the respective
homologue of the named markers in the organism which is
particularly in question for a method as disclosed herein. In
certain embodiments of the present invention, this organism is a
mammal and, in one embodiment, may be a human being. Thus, in one
embodiment of the present invention, the named markers shall be the
human homologues of the respective denominated ones.
[0036] Generally throughout the text, the term "(cell)
proliferation marker" or "marker for cell proliferation" in the
various grammatical forms is used to denominate proteins as well as
nucleic acid markers. In case the protein name of a marker such as
e.g. "replication protein" is used herein, this use shall be
understood metonymically and pertain as well to the protein as to
the nucleic acid marker molecules encoding the particular
protein
[0037] A marker useful according to the present invention may be
any molecule transcribed from a gene or any molecule translated
from such a transcript. Thus, "gene product", as used in the
context of the present invention, may comprise polynucleotides such
as, e.g., DNA or RNA and polypeptides such as proteins,
proteoglycans, peptides, etc. "Expression product(s)" as used in
the context of the present invention shall comprise any transcript
of a gene locus in forward or reverse direction including any
reading frames and splicing variants. "Expression products" as used
herein shall thus comprise any alternative products encoded by the
nucleic acids of a particular gene locus.
[0038] INK4a encoded gene-products, as used in the context of the
present invention, shall be any mRNA transcribed from the INK4a
gene locus or any polypeptide translated from such an mRNA. In one
embodiment of the invention, the expression products encoded by the
INK4a gene may exhibit molecular weights of about 5 to 40 kDa or
any value in between, and preferably of about 10 to 20 kDa or any
value in between, and most preferably of about 14 to about 19 kDa
or any value in between, respectively.
[0039] INK4a gene-products suitable for the method according to the
present invention may comprise, e.g., gene-products such as, e.g.,
p16.sup.INK4a and p14ARF.
[0040] The term "(cell) proliferation marker" or "marker for cell
proliferation", as used in the context of the present invention,
shall comprise any marker molecule known in the art to be
characteristic for the proliferation status of cells. The
proliferation status may be, e.g., a status of actively
proliferating cells, of retarded cell proliferation, of arrested
cell proliferation, of senescent cells, of terminally
differentiated cells, of apoptosis, etc. In one embodiment of the
invention, the cell proliferation marker is a marker molecule
characteristic for active cell proliferation. In another embodiment
of the invention, the proliferation marker molecule may be a
molecule characteristic for arrested, terminally differentiated,
senescent, or apoptotic cells.
[0041] In certain embodiments, proliferation markers for use in the
context of the present invention may comprise genes engaged in the
DNA replication, such as e.g., proteins of the pre-initiation
complex or of the replication fork. Such molecules may comprise,
e.g., helicases, such as eucaryotic helicase or MCM proteins (MCM2,
MCM3, MCM4, MOMS, MCM6, MCMI), protein TP as disclosed in WO0050451
and W00217947 (also denominated HELAD1, Pomfil2, Unc-53), kinases
or phosphatases engaged in the replication process such as, e.g.,
CDC6, CDC7 protein kinase, Dbf4, CDC14 protein phosphatase, CDC45
and MCM10. Furthermore, proliferation markers may comprise proteins
engaged in the processive replication fork such as, e.g., PCNA or
DNA polymerase delta, replication protein A (RPA), replication
factor C (RFC), and FEN1.
[0042] In other embodiments, the proliferation markers may comprise
molecules necessary for the maintenance of cell proliferation such
as Ki67, Ki-S5, or Ki-S2. In this embodiment, proteins may be
present, e.g., throughout the whole cell cycle. They are useful for
performing a method according to the present invention provided
they are characteristic of active cell proliferation and are not
significantly expressed in arrested, terminally differentiated,
apoptotic, or senescent states of cells. Ki67, Ki-S2, and Ki-S5 as
used herein shall denominate the protein marker molecules detected
by the respective antibodies as well as the nucleic acids encoding
these antigens.
[0043] In another embodiment, the cell proliferation markers for
use in a method according to the present invention may be a marker
molecule characteristic of retarded or ceased cell proliferation
such as, e.g., a senescence marker, a cell cycle arrest marker, a
marker characteristic for terminally differentiated cells, or an
apoptosis marker. Such molecules comprise, e.g., p21, p27,
Caspases, BAD, CD95, fas-ligand, parp-proteins, etc.
[0044] Discrimination as used in the context of the present
invention shall comprise an assessment whether a sample is to be
classified in one or another way. In one embodiment of the
invention, the discrimination pertains to the assessment of a
tissue, or components thereof, being dysplastic or being not
dysplastic. Thus, the discrimination as used herein is a judgement
about the growth properties of cells in a biological sample.
[0045] In one embodiment of the present invention, the
discrimination comprises the detection of expression of an INK4a
gene product simultaneously with the detection of expression of a
marker characteristic for active cell proliferation. In this case,
cells co-expressing both marker molecules are to be classified as
being dysplastic.
[0046] In another embodiment of the present invention, the
discrimination comprises the detection of an INK4a gene product
simultaneously with the detection of expression of a marker
characteristic for arrested, ceased, or retarded cell
proliferation. In this case, cells co-expressing both marker
molecules are to be classified as being non-dysplastic.
[0047] In certain embodiments of the present invention, it will be
useful to detect the presence or absence and/or the level of more
than two marker molecules. In one embodiment, one INK4a gene
expression product will be detected in concert with two or more
markers for cell proliferation. This may be useful to enhance the
identification of proliferation properties of the INK4a gene
product-expressing cells in samples. Some proliferation markers are
restricted to specific phases of the cell cycle or are present in
low abundance in cells. Due to this fact, in some cases, the
detection of proliferating cells expressing INK4a gene products may
be improved by detection of two or more proliferation markers. In
such cases, e.g., one proliferation being expressed during the
whole proliferative cell cycle may be detected simultaneously by
markers characteristic for specific cells cycle phases. For
example, Ki67, Ki-S5, or Ki-S2 may be detected together with mcm5,
mcm2, PCNA, rpA, rfC, etc. In other cases, e.g., proteins engaged
in the DNA replication may be detected together with Ki67, Ki-S5,
or Ki-S2. In yet another case, Ki67 may be detected together with
Ki-S2. It must be understood that these examples are intended to
exemplify combinatorial possibilities, and shall not be
comprehensive, so that various other combinations of proliferation
markers are likewise useful and suitable in the procedure of the
method according to the present invention.
[0048] As the case may be, combination of two or more cell
proliferation marker molecules may be applied in a method as
disclosed herein. In another embodiment, two or more marker
molecules detectable over larger stretches of the cell cycle or
even over the whole cell cycle, or in an actively proliferating
cell, may be detected in concert in a method as disclosed herein. A
combination of more than one cell proliferation marker molecules
may be useful generally for improving the sensitivity of the
detection of the proliferation characteristics of cells.
[0049] In certain embodiments of the present invention, a
combination may furthermore comprise other marker molecules such as
senescence marker molecules, markers for arrested cells, markers
for terminally differentiated cells, markers for apoptotic cells,
markers for viral infection or for viral activity in cells, or cell
cycle regulatory protein markers. In certain embodiments, in
connection with dysplasias being associated with HPV infection, a
detection of HPV associated marker molecules or markers for viral
activity may be of use for a detection of a dysplasia. The methods
useful for detection of HPV infection in samples are known to those
of skill in the art. These methods may comprise methods employing
probes specific for HPV agents or may employ nucleic acid
amplification reactions. The detection of a viral infection may be
carried out simultaneously or subsequently to the detection of the
INK4a and proliferation marker molecules.
[0050] Dysplastic, as used in the context of the present invention,
shall refer to dysplasias from mild to severe dysplasias and their
precursory stages, as well as carcinoma such as carcinomas in situ
or invasive carcinomas and disseminated tumor cells. Thus,
dysplastic as used herein shall also comprise early and precursory
stages of dysplasias and carcinomas.
[0051] Cells over-expressing INK4a gene products without being
dysplastic (non-dysplastic as used herein) as mentioned herein may
comprise, e.g., metaplastic cells, senescent cells, terminally
differentiated cells, or cells that in certain stages of the cell
cycle exhibit elevated levels of the INK4a gene products. In
certain cells, elevated levels of INK4a gene products may even be
effected as a response to external signals such as hormones,
transmitters, etc. In one embodiment of the present invention, the
non-dysplastic cells over-expressing INK4a gene products comprise,
e.g., metaplastic cells, endometrial cells, etc.
[0052] The method for detection of the expression level of the
INK4a encoded gene-products and/or the proliferation marker gene
products according to the present invention is any method, which
may (but need not) be, e.g., suited to detect even very small
amounts of specific biological molecules in biological samples. The
detection reaction according to the present invention is a
detection either on the level of nucleic acids or on the level of
polypeptides.
[0053] A marker molecule is said to be detectable as used in the
context of the present invention, provided the marker may be
detected in the course of suitable detection procedure such as,
e.g., in situ hybridization, immuno-chemical staining, hybrid
capture assay, etc. The level of expression of a marker molecule
may be made detectable using suitable reporter reactions such as,
e.g., a chromogenic or fluorescence based immuno-chemical staining
or in-situ-hybridization procedure for microscopic or automated
analysis. Suitable methods for enhancing the reporter signal known
to those of skill in the art may be applied in the course of a
method according to the present invention. Thus, the marker is said
to be detectable in a case where the staining supersedes the
respective background staining inherently obtained in the
immuno-chemical staining procedure so as to produce significant
staining results.
[0054] The marker molecules may be detected using reagents that
specifically recognize these molecules. The detection reaction for
the INK4a gene-products and/or the proliferation marker gene
products may comprise one or more reactions with detecting agents
either recognizing the initial marker molecules or recognizing the
prior molecules used to recognize other molecules.
[0055] In certain embodiments of the present invention, two or more
probes may be used for the detection of one single marker molecule.
For example, two or more different binding agents (e.g.,
antibodies) or oligonucleotide probes directed against one single
marker molecule (or, as the case may be, against different epitopes
or different sequences) may be used in the course of the method as
disclosed herein.
[0056] The detection of the different gene products may be
performed in one reaction vessel or container or in different
containers simultaneously or subsequently in time. Thus, the
different gene products may be detected simultaneously in one cell
co-expressing both products. Otherwise, cells co-expressing the
gene products may be used for separated detection reaction
(separated in space or in time) to detect each a single marker in
the cells. In another embodiment, there might be cells expressing
one or the other marker. The detection of the marker molecules in
the different cells may also be performed simultaneously or
separately in time and/or space.
[0057] The detection reaction further may comprise a reporter
reaction indicating the presence or absence and/or the level of the
marker molecule gene-products. The reporter reaction may be, for
example, a reaction producing a coloured compound, a
bioluminescence reaction, a fluorescence reaction, generally a
radiation emitting reaction, etc.
[0058] In certain embodiments, different marker molecules may be
recognized by agents that produce different reporter signals, so
that the signals referring to marker molecules can be
distinguished. In one preferred embodiment of the invention, the
detection of the expression of the two or more INK4a gene-products
and/or proliferation marker gene products is carried out
simultaneously. In this case, the reporter reaction may employ, for
example, different fluorescent labels for the different molecules
detected.
[0059] However, within the context of the present invention, it
must not necessarily be answered whether the one or the other
proliferation marker or INK4a marker gene product is expressed in
the cells. In certain embodiments, the question is whether any
proliferation marker and/or INK4a gene product is expressed. In the
course of the experiments, a procedure may be chosen that gives the
same fluorescence signal as an indication of the presence of a
proliferation marker. This procedure is suitable to improve
sensitivity of the detection of the cell proliferation
characteristics (different markers characteristic for active cell
proliferation). As the case may be, the procedure may be applied so
as to render one detectable signal for three, four, or even more
marker molecules characteristic of cell proliferation. Analogously,
the same may under certain circumstances be true for the INK4a gene
expression products. It must be understood that different staining
signals for different proliferation marker molecules may be
desirable. The procedures may be applied to the necessities of the
respective experiment.
[0060] In certain embodiments of the present invention, a
combination of one or more (e.g., two different) INK4a gene
products may be detected with a combination of one or more (e.g., a
set of two, a set of three, a set of four, a set of five or a set
of even more) markers for cells proliferation. In some cases, the
detection of the marker molecules for cell proliferation may render
only one reporter signal. In other cases, each single marker for
cell proliferation may render a specific reporter signal or groups
of marker molecules may render specific reporter signals.
[0061] Signals for the indication of the presence of
immunoreactivity may be chromogenic signals produced by various
methods known in the art. Alternatively, or even in combination,
fluorescent signals may be used. Suitable reporter signals comprise
fluorescent labels such as fluorescein, rhodamine, etc.
[0062] Applicable formats for the detection reaction according to
the present invention may be blotting techniques, such as
Western-Blot, Southern-blot, Northern-blot, and immunocytochemical,
or immunohistochemical procedures. The blotting techniques are
known to those of ordinary skill in the art, and may be performed
as, for example electro-blots, semidry-blots, vacuum-blots or
dot-blots. Immunocyto/histochemical staining procedures are known
to those of skill in the art, and may comprise binding
agent-mediated detection of polypeptides as well as in situ
hybridisation techniques. Both different techniques may even be
applied simultaneously. In certain embodiments, hybrid capture of
nucleic acids may be used for the detection. Amplification
reactions may also be applicable for the detection of e.g. nucleic
acid molecules.
[0063] In one embodiment of the invention, the detection of the
level of INK4a and/or proliferation marker gene-products is carried
out by detection of the respective nucleic acids (e.g., mRNA) or
fragments thereof present in the sample. The means for detection of
nucleic acid molecules are known to those skilled in the art. The
procedure for the detection of nucleic acids can be carried out by,
for example, a binding reaction of the molecule to be detected to
complementary nucleic acid probes, proteins with binding
specificity for the nucleic acids, or any other entities
specifically recognizing and binding to said nucleic acids. In one
embodiment, in situ hybridisation of oligonucleotide probes to
nucleic acids in a sample may be used for the detection of
expression products or markers.
[0064] A probe, as used in the context of the present invention,
may be any agent binding specifically to a molecule. In the case of
nucleic acids, a probe may be an oligonucleotide hybridising to a
particular sequence. In one embodiment, the probe may be, e.g., a
primer. In the case of the detection of polypeptides or proteins,
the probe as used herein may be, e.g., a binding agent such as an
antibody. In certain embodiments of the present invention, the
probes may be detectably labelled. The label may be selected from
the group comprising a radioisotope, a bioluminescent compound, a
chemiluminescent compound, a fluorescent compound, a metal chelate,
or an enzyme. Probes may be applied in any detection procedure
known in the art as, for example, in the course of an in situ
hybridisation procedure, in the course of hybrid capture assays, in
the course of immuno-chemical staining reaction, in the course of
blotting techniques, etc.
[0065] This method may be performed as well in vitro as directly in
situ for example, in the course of a detecting staining reaction.
Another way of detecting the marker mRNAs in a sample performed in
the method according to the present invention is an amplification
reaction of nucleic acids, which can be carried out in a
quantitative manner such as, for example, the polymerase chain
reaction. In a preferred embodiment of the present invention, real
time (RT) PCR may be used to quantify the level of marker mRNAs in
samples of dysplasias or tumors (cells or tissue samples).
[0066] In another preferred embodiment of the invention, the
detection of the level of INK4a and/or proliferation marker
gene-products is carried out by determining the level of expression
of a protein or fragments thereof. The determination of the marker
gene-product on the protein level can, for example, be carried out
in a reaction comprising a binding agent specific for the detection
of the particular marker polypeptide.
[0067] The binding agents can be used in many different detection
techniques including, for example, in western-blot, ELISA or
immuno-precipitation. Generally, polypeptide binding agent based
detection can be carried out as well in vitro as directly in situ
as, for example, in the course of an immuno-chemical staining
reaction. Any other method for determining the amount of particular
polypeptides in biological samples can be used according to the
present invention.
[0068] The immuno-cytochemical imaging procedures for use in the
context of the present invention may comprise, e.g., the staining
of cytological or histological preparations with chromogenic or
fluorescent dyes. The staining may comprise, e.g., binding of the
molecules to be detected by a first binding agent, which itself is
detected by a secondary binding agent which may be labelled. The
first binding agent may in certain embodiments be a nucleic acid or
a protein binding agent (e.g. an antibody) and the secondary
binding agent may be, e.g., a secondary antibody recognizing the
first binding agent.
[0069] Any methods known in the art for performing staining of
cytochemical or histochemical staining may be applied in the course
of a method according to the present invention.
[0070] Binding agents as used in the context of the present
invention for the detection of the level of INK4a polypeptides such
as p16.sup.INK4a or p14ARF polypeptides and proliferation marker
polypeptides such as, e.g., mcm5, mcm2, Ki67, Ki-S5, PCNA, or Ki-S2
polypeptides may comprise antibodies and antigen-binding fragments,
bi-functional hybrid antibodies, peptidomimetics containing minimal
antigen-binding epitopes, anti-cullines (anti-Caline.TM.), etc.
[0071] An antibody or antigen-binding agent is said to react
specifically if it reacts at a detectable level with a protein
disclosed herein, and does not significantly react with other
proteins. The antibodies according to the present invention may be
monoclonal or polyclonal antibodies. As used herein, the term
antibody or monoclonal antibody is meant to include intact
molecules as well as antibody fragments. Moreover, antibodies of
the present invention include chimeric, single chain, and humanized
antibodies.
[0072] According to the present invention binding agents may be
used isolated or in combination. By means of combination, it is
possible to achieve a higher degree of sensitivity. The term
antibody, preferably, relates to antibodies which consist
essentially of pooled monoclonal antibodies with different epitopic
specificities, as well as distinct monoclonal antibody
preparations.
[0073] Monoclonal antibodies are made from antigen-containing
fragments of the polypeptide of the invention using any of a
variety of techniques known to those of ordinary skill in the art
(see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, 1988). In one such technique, an
immunogen comprising the antigenic polypeptide, or a synthetic part
thereof, is initially injected into any of a wide variety of
mammals (e.g., mice, rats, rabbits, sheep, and goats). In this
step, the polypeptides of this invention may serve as the immunogen
without modification. Alternatively, particularly for relatively
short polypeptides, a superior immune response may be elicited if
the polypeptide is joined to a carrier protein, such as bovine
serum albumin or keyhole limpet hemocyanin. The immunogen is
injected into the animal host, preferably according to a
predetermined schedule incorporating one or more booster
immunizations, and the animals are bled periodically. Polyclonal
antibodies specific for the polypeptide may then be purified from
such antisera by, for example, affinity chromatography using the
polypeptide coupled to a suitable solid support.
[0074] Within the context of the present invention, it must not
necessarily be answered whether one or the other proliferation
marker is expressed in the cells. In certain embodiments, the main
question may be whether any proliferation marker is expressed. In
the course of the experiments, a procedure was chosen that gives
the same fluorescence signal as an indication of the presence of a
proliferation marker. This procedure is suitable to improve
sensitivity of the detection of the cell proliferation
characteristics. As the case may be, the procedure may be applied
so as to render one detectable signal for three, four, or even more
marker molecules characteristic for cell proliferation.
Analogously, under certain circumstances, the same may be true for
the INK4a gene expression products. it must be understood, that, as
the case may be, different staining signals for different
proliferation marker molecules may be desirable. The procedures may
be applied to the necessities of the respective experiment.
[0075] The INK4a gene-products and/or proliferation marker gene
products may, according to the present invention, be detected
simultaneously. In this context, simultaneously according to the
present invention shall mean either literally at the same instant
or within the same testing procedure, whereby the single detection
steps are temporarily consecutive.
[0076] The detection procedure according to the present invention
may, furthermore, comprise a cytochemical staining procedure
rendering a chromogenic or fluorescent staining of cells or cell
compartments. Such staining procedures are known to those of skill
in the art and may comprise, for example, staining for acidophilic
or basophilic structures, of subcellular regions (e.g. the nucleus,
the mitochondria, the golgi, the cytoplasm, etc.), of specific
molecules (the chormosomes, of lipids, of glycoproteins, of
polysaccharids, etc.) in the cytological specimens. Fluorescence
dyes such as DAPI, Quinacrin, Chromomycin, etc. may be employed.
Furthermore, chromogenic dyes such as Azan, Acridin-orange,
Hematoxylin, Eosin, Sudan-red, and Thiazin-stains (Toluidin-blue,
Thionin) may be applied. In other embodiments, staining procedures
such as Pap-staining, Giemsa-staining, Hematoxylin-Eosin staining,
van-Gieson staining, Schiff-staining (using Schiff reagent),
staining procedures employing precipitation of metals (such as,
e.g., of silver in staining procedures employing Silver Nitrate),
or insoluble stains such as, e.g., of Turnbulls-blue (or other
insoluble metal cyanides), etc. may be used in the course of a
method as disclosed herein. It must be understood, that the named
dyes and staining methods shall be examples for the applicable
methods, and that any other method known in the art may be applied
to a method as disclosed herein.
[0077] The staining procedures may produce chromogenic stains for
light microscopic inspection or fluorescent stains for inspection
under fluorescence microscopic conditions. In another embodiment of
the present invention, radiation emitting procedures, procedures
employing substances impairing the transmission of radiation, or
other contrast media for imaging of the cytological conditions in a
sample (e.g., the generation of optical impression by means such as
(micro-)autoradiographic or (micro-)radiographic picture
generation) may be of use for a method according to the present
invention.
[0078] All the staining and imaging procedures may be used for
analysis not only in microscopic procedures, but also in automated
analysis procedures such flow cytometry, automated microscopic
(computerized or computer aided) analysis, or any other method for
analysis of stained cytological specimens.
[0079] The analysis of the staining or imaging results of the
different procedures may be performed in a single analysis step or
in different subsequent steps. For example, the light microscopic
inspection of a specimen may be performed before or after
fluorescence microsopic inspection of the specimen. In fluorescence
microscopy, the analysis of different stains with different
excitation wavelengths may be analyzed simultaneously or
subsequently. Other imaging methods may be employed simultaneously
or subsequently to the named procedures.
[0080] There may be various circumstances, under which combinations
of different staining methods will be suitable. For example, in
cases where no satisfying cytological staining results may be
achieved by immuno-chemical staining, the additional application of
general cytological staining techniques may be suitable.
[0081] A sample according to the method of the present invention
may comprise any sample comprising cells. Samples may comprise,
e.g., secretions, smears, body fluids, and cell- or
tissue-samples.
[0082] In one embodiment of the present invention, samples comprise
cells of the anogenital tract, of the respiratory tract, or of the
skin and its appendages. In certain embodiments, the cells may be
cells of the uterine cervix, the vagina, the vulva, the penis, the
anus, the rectum, the bronchic tree, the lung, the peritoneum, the
peritoneal space, the naso-pharyngeal space, the oral cavity, or
the skin. In certain embodiments of the present invention, the
sample may be a histological sample, a biopsy, or a cytological
sample such as, e.g., a smear, a swab, a wash, or a body fluid
containing cells (sputum, a secretion, saliva, etc.). In certain
embodiments of the present invention, the samples may comprise
cells infected by papilloma virus. The samples may, in certain
embodiments, comprise cervical smears, bronchioalveolar lavages,
etc.
[0083] In certain special embodiments of the present invention, the
sample may be prepared as a monolayer or thin layer preparation of
a cytological specimen. The respective methods for preparation of
monolayer or thin-layer preparation in cytology are known to those
of skill in the art. In one embodiment, the preparation may
comprise, e.g., the ThinPrep technology. Other methods comprise
conventional smears, or method employing suspensions of cells for
preparation of the cytological specimens.
[0084] Preparation of a sample may comprise, e.g., obtaining a
sample of a tissue, of a body fluid, or of cells from a patient.
According to the present invention, preparation of the sample may
also comprise several steps of further preparations of the sample,
such as preparation of dissections, preparation of cell
suspensions, spreading or applying the cells to be examined onto
microscopic slides, preparation of tissue arrays, isolation of
polypeptides or nucleic acids, preparation of solid phase fixed
peptides or nucleic acids, or preparation of beads, membranes, or
slides to which the molecules to be determined are coupled
covalently or non-covalently.
[0085] In certain embodiments of the present invention, the method
may be performed in an automated manner. The automation of the
method may be achieved by automated staining and analysis of
histological or cytological specimens on a solid surface by
microscopic means. In another embodiment, the automation may
comprise a flow-cytometric analysis of the staining of cells in
solution.
[0086] The dysplastic lesions to which the method according to the
present invention may be applied comprise any dysplastic lesions
characterized by over-expression of INK4a gene products such as,
e.g., p16.sup.INK4a a or p14ARF. In certain embodiments, those
lesions are dysplasias associated with infections by papilloma
viruses such as, e.g., HPV. In one embodiment, the HPV may be a
high risk HPV subtype such as HPV16, HPV18, HPV31, HPV 33, HPV35,
HPV 39, HPV 45, HPV 51, HPV 52, HPV56, HPV 58, HPV 59, HPV 66, HPV
68, etc. In one embodiment, the dysplastic lesions that may be
detected according to the present invention comprise anogenital
lesions, lesions of the respiratory tract, lesions of the head and
the neck, or lesions of the skin and its appendages. Such lesions
may comprise dysplasias, e.g., of the anus or rectum, of the vulva,
the vagina, the cervix or the penis, of the bronchic tree, the
lung, the oral cavity, or the nasopharyngeal space.
[0087] Another aspect of the present invention is a testing kit for
performing the method according to the present invention. The kit
may be, for example, a diagnostic kit or a research kit.
[0088] A kit according to the present invention comprises at least
an agent suitable for detecting the INK4a gene-products.
[0089] Thus a kit according to present invention may comprise:
reagents for the detection of one or more INK4a gene-products;
reagents for the detection of one or more proliferation marker
gene-products; reagents and buffers commonly used for carrying out
the detection reaction, such as buffers, reporters reactants (dyes,
etc.), carrier substances, and others; INK4a gene product samples
for carrying out a positive control reaction; and proliferation
marker gene product samples for carrying out a positive control
reaction.
[0090] The reagents for the detection of the marker gene-products
may include any agent capable of binding to the marker
gene-products. Such reagents may include proteins, polypeptides,
nucleic acids, peptide nucleic acids, glycoproteins, proteoglycans,
polysaccharides, or lipids.
[0091] The INK4a gene-product and proliferation marker gene product
samples for carrying out a positive control may comprise, for
example, nucleic acids in applicable form, such as solution or
salt, peptides in applicable form, tissue section samples, or
positive cells.
[0092] In a preferred embodiment of the invention, the detection of
the marker gene-products is carried out on the level of
polypeptides. In this embodiment, the binding agents may be, for
example, antibodies specific for the marker gene-products or
fragments thereof.
[0093] In another embodiment of the test kit, the detection of the
marker gene-products is carried out on the nucleic acid level. In
this embodiment of the invention, the reagent for the detection may
be, for example, a nucleic acid probe or a primer
reverse-complementary to said marker nucleic acids.
[0094] The present invention provides a method for the
discrimination of neoplastic and/or dysplastic and pre-neoplastic
lesions, identifiable by assessment of the over-expression of
p16.sup.INK4a, from other cells , which also detectably express
p16.sup.INK4a, in the course of histological and/or cytological
testing procedures. The method is based on the detection of
expressed gene-products of two or more INK4a gene products.
[0095] Thus the problem to be solved was to provide a method for
discrimination between dysplastic cells and other cells lacking
malignant growth potential. The method may be applied to any stage
of dysplasias and may be especially useful in early stages, when
cytological diagnostic methods based on the p16.sup.INK4a
over-expression need further information for the identification of
metaplastic cells.
[0096] Furthermore the present invention provides a kit for
performing the method according to the present invention.
EXAMPLES
[0097] The following examples are given for the purpose of
illustration only and are not intended to limit the scope of the
invention disclosed herein. For the purpose of illustration, the
methods disclosed herein are exemplified using histological
preparations. The histological examples aid to judge whether the
cells stained in one or the other way are to be classified as
dysplastic or metaplastic. The methods may easily be transferred to
cytological specimens by altering the protocol in appropriate
manner. These alterations are known to those of ordinary skill in
the art.
Example 1
Immunofluorescent Detection of the Over-Expression of p16.sup.INK4a
and Ki67 in Samples of the Uterine Cervix (Double Staining)
[0098] Sections of formalin fixed, paraffin embedded tissue samples
of the cervix uteri were immunofluorescent stained using antibodies
specific for p16.sup.INK4a and Ki67.
[0099] The tissue sections were rehydrated through incubation in
xylene and graded ethanol, and transferred to Aqua bidest. Antigen
retrieval was carried out with 10 mM citrate buffer (pH 6.0) for
p16.sup.INK4a and Ki67. Therefore, the slides were heated in a
waterbath for 40 min at 95-98.degree. C. The slides were cooled
down to RT for 20 minutes, and transferred to washing buffer (50 mM
Tris-HCl, 150 mM NaCl, 0.05% Tween 20/DakoCytomation: code no.:
S3006).
[0100] To avoid non-specific binding of the secondary antibody
(species: goat), the specimens were incubated with 10% goat serum
for 30 min at RT.
[0101] The slides were then incubated with the primary antibodies,
mouse anti-human p16.sup.INK4a antibody (3.48 .mu.g/ml) and rabbit
anti Ki67 (1:25) for 30 min at RT, after which the slides were
rinsed with washing buffer and placed in a fresh buffer bath for 5
min. Excess buffer was tapped off, and each specimen was covered
with 200 .mu.l of the secondary reagent containing goat anti-mouse
antibody, AlexaFluor.TM. 488 conjugated and goat anti-rabbit
antibody, and Alexa Fluo.TM. 546 conjugated, and then incubated for
30 min at RT. Then slides were washed two times as before and
directly mounted with a special mounting medium for
fluorescence.
[0102] Microscopic examination of the slides revealed that cells
immunoreactive with p16.sup.INK4a and also immunoreactive for Ki67
were only found in samples microscopically identified as samples of
dysplastic lesions. Cells stained by the p16.sup.INK4a specific
reaction originating from metaplasias were not stained by the
reaction specific for Ki67. Microscopic inspection of the cell
proliferation marker staining showed that metaplastic cells
over-expressing p16.sup.INK4a were not immunoreactive with the
antibodies directed against Ki67. Samples containing dysplastic
tissue areas, in contrast, comprised cells that were immunoreactive
with KI67 and with antibodies directed against p16.sup.INK4a. So,
in contrast to dysplasias in metaplasias, no cells were double
stained using the Ki67 and p16.sup.INK4a specific antibodies.
[0103] FIGS. 1-6 show the staining results for a severe dysplasia
of the cervix uteri and for a squamous metaplasia using antibodies
directed against Ki67 and against p16.sup.INK4a. Immunoreactivity
for Ki67 rendered red fluorescence, immunoreactivity for
p16.sup.INK4a rendered green fluorescence, and overlay of red and
green fluorescence rendered yellow fluorescence. In the dysplastic
specimen (FIGS. 1-3), many cells of the dysplasia showed nuclear
positivity for Ki67, as well as positivity for p16.sup.INK4a, and
thus gave rise to yellow fluorescence (see FIG. 3). In contrast, in
the metaplastic specimen (FIGS. 4-6), areas that positively
expressed p16.sup.INK4a did not show any positive staining for
Ki67, indicating lack of expression of the antigen in these areas.
No double staining was observed in this specimen (FIG. 6) and thus,
no cells in the specimen gave rise to yellow fluorescence.
[0104] These results show that the double staining of cells with
reagents specific for Ki67 allows discrimination of p16.sup.INK4a
over-expressing metaplasias from dysplasias.
Example 2
Detection of Cells Co-Expressing p14ARF and mcm2 in Samples of the
Uterine Cervix by In Situ Hybridization
[0105] Smears of the uterine cervix may be semi-quantitatively
analysed for the mRNA level of p16.sup.INK4a and mcm2 in an in situ
staining reaction. The staining reaction is performed as
follows:
[0106] For rehydration, the spray-fixed smears are incubated in
fresh 50% EtOH on a rocking device. The PEG film produced by the
fixation procedure is removed by intensive rinsing. Then, the
smears are rinsed in Aqua bidest. The smears are incubated with
proteinese K (10 .mu.g/ml in PBS) for 10 min at 37.degree. C. Then,
the slides are transferred to washing buffer (PBS/0.1% Tween20) and
finally, the area containing the cells is surrounded with a
lipid-pencil.
[0107] The hybridization mixture is prepared by mixing 50 .mu.l of
ready-to-use hybridization buffer (DAKO A/S, Glostrup, Danmark)
with about 5-10 pmol of the probes. The probes are biotin- and
Digoxygenin-labelled oligonucleotides of sequences complementary to
the respective mRNAs.
[0108] The hybridization mixture is heated to 95.degree. C. and
afterwards, equilibrated to 37.degree. C. After the boiling
procedure, the smears are incubated with each 50 .mu.l of the
hybridization mixture for 4 hours at 42.degree. C. The samples are
washed in excess volumes of the wash buffers two times in 2.times.
SSC at 37.degree. C. for 15 min, and once in 1.times. SSC at
37.degree. C. for 15 min. Then, the smears are rinsed two times at
room temperature in 2.times. SSC. Following this washing procedure,
the dissections are incubated for 30 min with blocking buffer (NEN,
blocking buffer) at room temperature, followed by a 1 hour
incubation with a 1:100 diluted (in blocking buffer, see above)
Streptavidin-alkaline phosphatase and monoclonal mouse HRP-labeled
anti-Digoxygenine antibodies (Molecular Probes). The smears are
then washed 2 times in 1.times. PBS/0.1% Triton X-100 for 10 min at
room temperature, followed by one wash step with 1.times. PBS, 50
mM MgCl.sub.2 (pH 9.2) for 10 min at room temperature. Then the
staining reaction is performed with ELF 97 phosphate (Molecular
Probes) for 10 sec to 7 min at room temperature. Excess substrate
is washed 3 times with 1.times. PBS/0.1% Triton X-100 for 10 min at
room temperature. In a second staining step, the section is
incubated with Tyramides-Alexa-Fluor 594 for 10 sec to 7 min.
Excess substrate is washed 3 times with 1.times. PBS/0.1% Triton
X-100 for 10 min at room temperature. Finally the smears are dipped
in H.sub.2O.sub.dest and embedded with Fluorescence mounting medium
(DakoCytomation). Then the stained dissections can be analysed by
fluorescence microscopy.
[0109] Microscopic examination of the slides reveals that cells
positive for expression of p16.sup.INK4a and mcm2 are only found in
samples that are microscopically identified as samples of
dysplastic lesions. Cells stained by the p16.sup.INK4a specific
reaction that are identifiable as metaplasias are not stained by
the reaction specific for mcm2. The microscopic inspection of the
mRNA hybridization shows that metaplastic cells over-expressing
p16.sup.INK4a do not significantly express mRNA of mcm2. Dysplastic
cells, in contrast, are stained by in situ hybridization with
probes specific for mcm2, and with probes directed against
p16.sup.INK4a. So, in contrast to dysplastic cells, in metaplastic
cells, no double staining using the mcm2 and p16.sup.INK4a specific
probes is observed.
[0110] These results show that the double staining of cells with
reagents specific for mcm2 allows discrimination of p16.sup.INK4a
over-expressing metaplasias from dysplasias.
Example 3
Immunofluorescent Detection of the Over-Expression of p16.sup.INK4a
and Ki-S2 in Samples of the Uterine Cervix (Double Staining)
[0111] Merckofix.TM. fixed cytological samples (conventional smears
and liquid-based cytology (ThinPreps.TM.)) of the cervix uteri are
immunofluorescent stained using antibodies specific for
p16.sup.INK4a and Ki-S2.
[0112] Conventional smears and liquid based cytological samples
(ThinPreps.TM.) are rehydrated in ethanol (50%) for 10 min and
transferred in Aqua bidest. Antigen retrieval is carried out with
10 mM citrate buffer (pH 6.0) for p16.sup.INK4a and Ki67.
Therefore, the slides are heated in a waterbath for 40 min at
95.degree.-98.degree. C. The slides are cooled down to RT for 20
minutes, transferred to washing buffer (50 mM Tris-HCl, 150 mM
NaCl, 0.05% Tween 20/DakoCytomation: code no.: S3006), and finally,
the samples are surrounded with a lipid-pencil.
[0113] To avoid non-specific binding of the secondary antibody
(species: goat) the specimens are incubated with 10% goat serum for
30 min at RT.
[0114] The slides are then incubated with the primary antibodies,
mouse anti-human p16.sup.INK4a antibody (clone E6H4) (3.48
.mu.g/ml) and rabbit anti-Ki-S2 (1:25) for 30 min at RT, and then
the slides are rinsed with washing buffer and placed in a fresh
buffer bath for 5 min. Excess buffer is tapped off, and the
specimen is covered with 200 .mu.l of the secondary reagent
containing goat anti-mouse antibody, AlexaFluor.TM. 488 conjugated
and goat anti rabbit antibody, and Alexa Fluor.TM. 546 conjugated
and then incubated for 30 min at RT. Then, slides are washed two
times as before and directly mounted with a special mounting medium
for fluorescence.
[0115] Microscopic examination of the slides reveals that cells
immunoreactive with p16.sup.INK4a and Ki-S2 may be identified
microscopically as dysplastic cells. Cells stained by the
p16.sup.INK4a specific reaction, which are not stained by the
reaction specific for Ki-S2, may be classified by an experienced
pathologist as either being metaplastic or of endometrial origin.
Microscopic inspection of the cell proliferation marker staining
shows that metaplastic cells over-expressing p16.sup.INK4a are not
immunoreactive with the antibodies directed against Ki-S2.
Dysplastic cells, in contrast, are immunoreactive with Ki-S2 and
with antibodies directed against p16.sup.INK4a. So, in contrast to
dysplastic cells, in metaplasias, no cells are double stained using
the Ki-S2 and p16.sup.INK4a specific antibodies.
[0116] These results show that double staining of cells with
reagents specific for Ki-S2 allows discrimination of p16.sup.INK4a
over-expressing metaplastic cells from dysplastic cells.
Example 4
Immunofluorescent Detection of the Over-Expression of
p16.sup.INK4a, Ki67 and PCNA in Bronchioalveolar-Lavage Samples of
Individuals with Diagnosed Small Cell Lung Cancer (Double
Staining)
[0117] Cells contained in bronchioalveolar lavage specimens of
patients are prepared according to ThinPrep technology.
Merckofix.TM.-fixed cytological samples of the lavages of patients
diagnosed with small cell lung cancer are immunofluorescent stained
using antibodies specific for p16.sup.INK4a, Ki67, and PCNA.
[0118] In this experiment, a procedure is used that does not
discriminate between staining originating from immunoreactivity
against the two proliferation markers PCNA and Ki67. Within the
context of the present invention, whether one or the other
proliferation marker is expressed in the cells must not necessarily
be answered. The main question is whether any proliferation marker
is expressed. In the course of the experiments, a procedure is
chosen that gives the same fluorescence signal as an indication of
the presence of a proliferation marker. This procedure is suitable
to improve sensitivity of the detection of the cell proliferation
characteristics. As necessary, the procedure may be applied so as
to render one detectable signal for three, four, or even more
marker molecules characteristic of cell proliferation. Analogously,
the same may under certain circumstances be true for the INK4a gene
expression products. It must be understood that different staining
signals for different proliferation marker molecules may be
desirable. The procedures may be applied to the necessities of the
respective experiment.
[0119] The tissue sections are rehydrated through incubation in
xylene and graded ethanol, and then transferred to Aqua bidest.
Conventional smears and liquid-based cytological samples
(ThinPreps.TM.) are rehydrated in ethanol (50%) for 10 min, and
transferred in Aqua bidest. Antigen retrieval is carried out with
10 mM citrate buffer (pH 6.0) for p16.sup.INK4a, Ki67, and PCNA.
Therefore, the slides are heated in a waterbath for 40 min at
95-98.degree. C. The slides are cooled down to RT for 20 minutes,
transferred to washing buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05%
Tween 20/DakoCytomation: code no.: S3006), and finally, the samples
are surrounded with a lipid-pencil.
[0120] To avoid non-specific binding of the secondary antibody
(species: goat), the specimens are incubated with 10% goat serum
for 30 min at RT.
[0121] The slides are then incubated with the primary antibodies,
mouse anti-human p16.sup.INK4a antibody (3.48 .mu.g/ml), rabbit
anti-Ki67, and rabbit anti-PCNA (each 1:25) for 30 min at RT. The
slides are then rinsed with washing buffer and placed in a fresh
buffer bath for 5 min. Excess buffer is tapped off, and the
specimen is covered with 200 .mu.l of the secondary reagent
(containing goat anti-mouse antibody, AlexaFluor.TM. 488 conjugated
and goat anti rabbit antibody and, Alexa Fluor.TM. 546 conjugated)
and then incubated for 30 min at RT. Then slides are washed two
times as before and directly mounted with a special mounting medium
for fluorescence.
[0122] Microscopic examination of the slides reveals that cells
immunoreactive with p16.sup.INK4a and also with Ki67 or PCNA may be
identified microscopically as cells of small cell lung cancer.
Cells stained by the p16.sup.INK4a a specific reaction, originating
from metaplasias, are not stained by the reaction specific for Ki67
and PCNA. The microscopic inspection of the cell proliferation
marker staining shows that metaplastic cells over-expressing
p16.sup.INK4a are not immunoreactive with antibodies directed
against Ki67 and PCNA. Samples containing dysplastic cells, in
contrast, comprise cells, that are immunoreactive with Ki67/PCNA
and with antibodies directed against p16.sup.INK4a. So, in contrast
to dysplasias, in metaplasias, no cells may be triple-stained using
the Ki67, PCNA, and p16.sup.INK4a specific antibodies.
[0123] These results show that the triple staining of cells with
reagents specific for Ki67/PCNA allows discrimination of
p16.sup.INK4a over-expressing non-dysplastic cells from
dysplasias.
Example 5
Flow Cytometric Detection of Dysplastic Cells by Simultaneous
Detection of mcm5 mRNA, p14ARF Protein, and Ki67 Protein in Cells
of Cervical Origin
[0124] Cytological samples (Cell suspensions in PBS, pH 7.4) of the
cervix uteri were fluorescent stained using antibodies specific for
p14ARF and Ki-67 and oligoprobes for mcm-5 and evaluated by
three-color fluorescent FACS analysis.
[0125] Cells were centrifuged, the supernatant decanted and fixed
and permeabilized with 100 ml Permeafix (Ortho Diagnostic, Raitan,
N.J., USA) for 1 hour at room temperature. Cells were washed in
sterile PBS, pH 7.4, pelleted, and re-suspended in 100 ml Permeafix
for 1 hour at room temperature.
[0126] Cells were washed in sterile PBS, pH 7.4, pelleted, and
re-suspended in sterile PBS. They were incubated with PE-conjugated
anti-p14ARF antibody and PE-Cy5-conjugated anti-Ki67 antibody for 1
h at +4.degree. C. Cells were washed in sterile PBS, pH 7.4,
pelleted, and re-suspended in 100 ml Permeafix for 30 min at room
temperature. Cells were washed in sterile PBS, pelleted by
centrifugation, and then washed again in 2.times. standard saline
citrate (SSC). After centrifugation, the cell pellet was
resuspended in hybridization solution (2.times. SSC, 30% formamide,
sonicated salmon sperm, and yeast transfer DNA) containing 500 ng
of 5-carboxy-fluorescein double end-labelled, mcm5-specific
oligonucleotides probes. The intercellular hybridization was
performed at 42.degree. C. for 1 hour, followed by successive
washes in 2.times. SSC, 0.5% Triton X-100, and 1.times. SSC, 0.5%
Triton X-100 at 42.degree. C. The cells were re-suspended for
analysis in PBS, pH 8.3, and analyzed on a flow cytometer (FACScan,
Becton Dickinson, IS). For each analysis, 30.000-100.000 gated
events were collected. Data analysis was performed using CellQuest
(Becton Dickinson, IS).
[0127] The flow cytometer analysis revealed that cells
immunoreactive with p14ARF and Ki-67, and/or reactive for the
oligoprobes of mcm5, could be identified only in samples of
patients with dysplastic lesions of the cervix. Samples from women
with no dysplastic lesions showed no concomitant staining of p14ARF
with Ki67 or mcm5.
[0128] These results show that the double-or-triple staining of
cells with reagents specific for Ki67 and/or mcm5 allows
discrimination of p14ARF over-expressing non-dysplastic cells from
p14ARF over-expressing dysplastic cells.
Example 6
Immunoenzymatic Detection of the Over-Expression of p16.sup.INK4a
and Ki67 in Histological Samples of the Uterine Cervix (Sequential
Double Staining)
[0129] Sections of formalin-fixed, paraffin-embedded tissue samples
of the cervix uteri were immunoenzymatically double-stained using
antibodies specific for p16.sup.INK4a and Ki67.
[0130] The tissue sections were rehydrated through incubation in
xylene and graded ethanol, and transferred to Aqua bidest, Antigen
retrieval was carried out with 10 mM citrate buffer (pH 6.0) for
p16.sup.INK4a and Ki67. Therefore, the slides were heated in a
waterbath for 40 min at 95-98.degree. C. The slides were cooled
down to RT for 20 minutes and transferred to washing buffer
(DakoCytomation).
[0131] Endogenous peroxidase activities were blocked with 3%
H.sub.2O.sub.2 (DakoCytomation) for 5 min at RT.
[0132] After washing the slides for 5 min at RT, they were
incubated with the first primary antibody, mouse anti-human
p16.sup.INK4a antibody (MTM) for 30 min at RT, and were then rinsed
with washing buffer and placed in a fresh buffer bath for 5 min.
Excess buffer was tapped off and each specimen was covered with 200
.mu.l of the secondary reagent (EnVision goat anti
mouse--Peroxidase/DakoCytomation), and incubated for 30 min at RT.
Then slides were washed three times as before. For the chromogenic
visualization, DAB (DakoCytomation) was used by incubating the
slides with the substrate chromogen complex for 10 min at RT. The
reaction was stopped in deionized water, and the slides placed in
wash buffer.
[0133] After washing, the slides were incubated with the second
primary antibody, rabbit anti-human Ki67 antibody (Dianova, clone
Ab-3) for 30 min at RT, and were then rinsed with washing buffer
and placed in a fresh buffer bath for 5 min. Excess buffer was
tapped off, and each specimen was covered with 200 .mu.l of the
secondary reagent (goat anti rabbit--Alkaline Phosphatase
labeled/DakoCytomation) and incubated for 30 min at RT. Then slides
were washed three times as before. For the chromogenic
visualization, FastRed (BioGenex) was used by incubating the slides
with the substrate chromogen complex for 30 min at RT. The reaction
was stopped in deionized water.
[0134] After counterstaining with hematoxylin (DakoCytomation) for
2 min at RT, the slides were incubated in running tap water for 10
min at RT, and then mounted with aqueous mounting medium
(Aquatex/MERCK).
[0135] The microscopic examination of the slides revealed that
cells immunoreactive with p16.sup.INK4a and Ki67 were found only in
samples that were identified microscopically as samples of
dysplastic lesions. Cells stained by the p16.sup.INK4a-specific
reaction, originating from metaplasias, were not stained by the
reaction specific for Ki67. Microscopic inspection of the cell
proliferation marker staining showed that metaplastic cells
over-expressing p16.sup.INK4a were not immunoreactive with
antibodies directed against Ki67. Samples containing dysplastic
tissue areas, in contrast, comprised cells that were immunoreactive
with Ki67 and with antibodies directed against p16.sup.INK4a. So,
in contrast to dysplasias, in metaplasias, no cells were
double-stained using the Ki67 and p16.sup.INK4a specific
antibodies.
[0136] These results (FIG. 7) show that double staining of cells
with reagents specific for Ki67 and p16.sup.INK4a also produces a
specific double-staining pattern in chromogenic staining
procedures.
Example 7
Immunoenzymatic Detection of the Over-Expression of p16.sup.INK4a
and Ki67 in Cytological Samples of the Uterine Cervix (Sequential
Double Staining)
[0137] Merckofix.TM. fixed cytological samples (conventional smears
and liquid-based cytology (ThinPreps.TM.)) of the cervix uteri were
immunoenzymatically double-stained using antibodies specific for
p16.sup.INK4a and Ki67.
[0138] Conventional smears and liquid-based cytological samples
were rehydrated in ethanol (50%) for 10 min at RT and transferred
in Aqua bidest. Antigen retrieval was carried out with 10 mM
citrate buffer (pH 6.0) for p16.sup.INK4a and Ki67. Therefore, the
slides were heated in a waterbath for 40 min at 95-98.degree. C.
The slides were cooled down to RT for 20 minutes and transferred to
washing buffer.
[0139] Endogenous peroxidase activities were blocked with 3%
H.sub.2O.sub.2 for 5 min at RT.
[0140] After washing, the slides were incubated with the first
primary antibody, mouse anti-human p16.sup.INK4a antibody, for 30
min at RT, and were then rinsed with washing buffer and placed in a
fresh buffer bath for 5 min. Excess buffer was tapped off, and each
specimen was covered with 200 .mu.l of the secondary reagent
(EnVision goat anti mouse--Peroxidase) and incubated for 30 min at
RT. Then the slides were washed three times as before. For the
chromogenic visualization, DAB (DakoCytomation) was used by
incubating the slides with the substrate chromogen complex for 10
min at RT. The reaction was stopped in deionized water, and the
slides placed in wash buffer.
[0141] After washing, the slides were incubated with the second
primary antibody, rabbit anti-human Ki67 antibody (Dianova, clone
Ab-3) for 30 min at RT, and were then rinsed with washing buffer
and placed in a fresh buffer bath for 5 min. Excess buffer was
tapped off, and each specimen was covered with 200 .mu.l of the
secondary reagent (goat anti rabbit--Alkaline Phosphatase
labeled/DakoCytomation) and incubated for 30 min at RT. Then the
slides were washed three times as before. For the chromogenic
visualization, Fast Red (BioGenex) was used by incubating the
slides with the substrate chromogen complex for 30 min at RT. The
reaction was stopped in deionized water.
[0142] After counterstaining with hematoxylin (DakoCytomation) for
2 min at RT, the slides were incubated in running tap water for 10
min at RT, and then mounted with aqueous mounting medium
(Aquatex/MERCK).
[0143] Microscopic examination of the slides revealed that cells
immunoreactive with p16.sup.INK4a l and Ki67 could be identified as
dysplastic cells on the basis of their morphology. Cells stained by
the p16.sup.INK4a. specific reaction originating from metaplasias
were not stained by the reaction specific for Ki67. The microscopic
inspection of the cell proliferation marker staining showed that
metaplastic cells over-expressing p16.sup.INK4a were not
immunoreactive with the antibodies directed against Ki67.
Dysplastic cells, in contrast, were immunoreactive with Ki67 and
with antibodies directed against p16.sup.INK4a. So, in contrast to
metaplastic cells, in dysplastic cells, double-staining of single
cells could be produced using the Ki67 and p16.sup.INK4a specific
antibodies.
[0144] These results (FIG. 8) show that the double staining of
cells with reagents specific for Ki67 and p16.sup.INK4a also
produces a specific double staining pattern in chromogenic staining
procedures.
* * * * *