U.S. patent application number 12/976428 was filed with the patent office on 2011-04-28 for assessment of human papilloma virus-related disease.
This patent application is currently assigned to QIAGEN GAITHERSBURG INC.. Invention is credited to James G. Lazar, Attila T. LORINCZ.
Application Number | 20110097708 12/976428 |
Document ID | / |
Family ID | 27371534 |
Filed Date | 2011-04-28 |
United States Patent
Application |
20110097708 |
Kind Code |
A1 |
LORINCZ; Attila T. ; et
al. |
April 28, 2011 |
ASSESSMENT OF HUMAN PAPILLOMA VIRUS-RELATED DISEASE
Abstract
This invention provides novel methods for assessing HPV
infection. Gene expression levels are used to assess the
progression of HPV infection from benign to malignant growth. Also
provided are kits for carrying out the methods of this
invention.
Inventors: |
LORINCZ; Attila T.; (North
Potomac, MD) ; Lazar; James G.; (Bethesda,
MD) |
Assignee: |
QIAGEN GAITHERSBURG INC.
Gaithersburg
MD
|
Family ID: |
27371534 |
Appl. No.: |
12/976428 |
Filed: |
December 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11681539 |
Mar 2, 2007 |
7879546 |
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12976428 |
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09970477 |
Oct 4, 2001 |
7291455 |
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11681539 |
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09210168 |
Dec 11, 1998 |
6355424 |
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09970477 |
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60082167 |
Apr 17, 1998 |
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60070486 |
Jan 5, 1998 |
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60069426 |
Dec 12, 1997 |
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Current U.S.
Class: |
435/5 |
Current CPC
Class: |
C12Q 2600/118 20130101;
C12Q 1/6806 20130101; C12Q 1/708 20130101; C12Q 1/6886 20130101;
C12Q 1/708 20130101; A01N 1/021 20130101; C12Q 2600/112 20130101;
C12Q 2523/101 20130101; C12Q 1/6806 20130101; C12Q 2543/10
20130101; C12Q 2545/114 20130101 |
Class at
Publication: |
435/5 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70 |
Claims
1. A method of determining the medical character of an HPV-based
disease comprising measuring the expression level of an HPV gene
selected from the group consisting of E6, E7, E2, and L1 and
correlating the expression level of the gene to the medical
character of the HPV-based disease.
2. The method of claim 1 wherein low expression of E6 and/or E7
indicates a benign HPV infection.
3. The method of claim 1 wherein low to medium expression of E6
and/or E7 indicates a benign HPV infection characterized as CIN
I.
4. The method of claim 1 wherein high expression of E6 and/or E7
indicates a neoplastic HPV infection.
5. The method of claim 1 wherein medium to high expression of E6
and/or E7 indicates a neoplastic HPV infection.
6. The method of claim 1 wherein high expression of E2 and/or L1
indicates a benign HPV infection.
7. The method of claim 1 wherein low to high expression of E2
indicates a benign HPV infection.
8. The method of claim 1 wherein medium to high expression of L1
indicates a benign HPV infection characterized as CIN I.
9. The method of claim 1 wherein low to undetectable expression of
E2 indicates a neoplastic HPV infection.
10. The method of claim 1 wherein low to medium expression of E6
and/or E7 and medium to high expression of L1 indicates a benign
HPV infection characterized as CIN I.
11. The method of claim 1 wherein low to medium expression of E6
and/or E7 and low to high expression of E2 indicates a benign HPV
infection characterized as CIN I.
12. The method of claim 1 wherein medium to high expression of E6
and/or E7 and low to undetectable expression of E2 and/or L1
indicates a neoplastic HPV infection characterized as CIN II/III or
cancer.
13. A method of determining progression of an HPV-based disease
comprising measuring the expression level of an HPV gene selected
from the group consisting of E6, E7, E2, and L1 at a first time
point and a second later time point, and correlating the change in
expression level to the medical character of the HPV-based
disease.
14. The method of claim 13 wherein an increase in E6 and/or E7 gene
expression from the first time point to the second time point
indicates progression of the HPV-based disease.
15. The method of claim 13 wherein a decrease in E6 and/or E7 gene
expression from the first time point to the second time point
indicates regression of the HPV-based disease.
16. The method of claim 13 wherein a change in E6 and/or E7 gene
expression from low at the first time point to high at the second
time point to indicates progression from a benign to a neoplastic
HPV infection.
17. The method of claim 13 wherein a change in E6 and/or E7 gene
expression from high at the first time point to low at the second
time point indicates regression from a neoplastic to a benign HPV
infection.
18. The method of claim 13 wherein a change in E6 and/or E7 gene
expression from low at the first time point to a range of medium to
high at the second time point indicates progression from a benign
to a neoplastic HPV infection.
19. The method of claim 13 wherein a change in E6 and/or E7 gene
expression from a range of medium to high at the first time point
to low at the second time point indicates regression from a
neoplastic to a benign HPV infection.
20. The method of claim 13 wherein a change in E6 and/or E7 gene
expression from a range of low to medium at the first time point to
a range of medium to high at the second time point indicates
progression from a benign to a neoplastic HPV infection.
21. The method of claim 13 wherein a change in E6 and/or E7 gene
expression from a range of medium to high at the first time point
to a range of low to medium at the second time point indicates
regression from a neoplastic to a benign HPV infection.
22. The method of claim 13 wherein a decrease in E2 and/or L1 gene
expression from the first time point to the second time point
indicates progression of the HPV-based disease.
23. The method of claim 13 wherein an increase in E2 and/or L1 gene
expression from the first time point to the second time point
indicates regression of the HPV-based disease.
24. The method of claim 13 wherein a change in E2 gene expression
from a range of low to high at the first time point to a range of
low to undetectable at the second time point indicates progression
from a benign to a neoplastic HPV infection.
25. The method of claim 13 wherein a change in E2 gene expression
from a range of low to undetectable at the first time point to a
range of low to high at the second time point indicates regression
from a neoplastic to a benign HPV infection.
26. The method of claim 13 wherein a change in E2 gene expression
from high at the first time point to undetectable at the second
time point indicates progression from a benign to a neoplastic HPV
infection.
27. The method of claim 13 wherein a change in E2 gene expression
from undetectable at the first time point to high at the second
time point indicates regression from a neoplastic to a benign HPV
infection.
28. The method of claim 13 wherein a change in L1 gene expression
from a range of medium to high at the first time point to a range
of low to undetectable at the second time point indicates
progression from a benign to a neoplastic HPV infection.
29. The method of claim 13 wherein a change in L1 gene expression
from a range of low to undetectable at the first time point to a
range of medium to high at the second time point indicates
regression from a neoplastic to a benign HPV infection.
30. The method of claim 13 wherein progression of the HPV-based
disease is indicated by: (a) an increase in E6 and/or E7 gene
expression from the first time point to the second time point; and
(b) a decrease in E2 and/or L1 gene expression from the first time
point to the second time point.
31. The method of claim 13 wherein regression of the HPV-based
disease is indicated by: (a) a decrease in E6 and/or E7 gene
expression from the first time point to the second time point; (b)
an increase in E2 and/or L1 gene expression from the first time
point to the second time point.
32. The method of claim 13 wherein progression from a benign to a
neoplastic HPV infection is indicated by: (a) an increase in E6
and/or E7 gene expression from low at the first time point to high
at the second time point; and (b) a decrease in E2 gene expression
from high at the first time point to undetectable at the second
time point.
33. The method of claim 13 wherein progression from a benign to a
neoplastic HPV infection is indicated by: (a) an increase in E6
and/or E7 gene expression from low at the first time point to high
at the second time point; and (b) a decrease in L1 gene expression
from a range of medium to high at the first time point to a range
of low to undetectable at the second time point.
34. The method of claim 13 wherein progression from a benign to a
neoplastic HPV infection is indicated by: (a) an increase in E6
and/or E7 gene expression from low at the first time point to high
at the second time point; (b) a decrease in E2 gene expression from
high at the first time point to undetectable at the second time
point; and (c) a decrease in L1 gene expression from a range of
medium to high at the first time point to a range of low to
undetectable at the second time point.
35. The method of claim 13 wherein regression from a neoplastic to
a benign HPV infection is indicated by: (a) a decrease in E6 and/or
E7 gene expression from high at the first time point to low at the
second time point; and (b) an increase in E2 gene expression from
undetectable at the first time point to high at the second time
point.
36. The method of claim 13 wherein regression from a neoplastic to
a benign HPV infection is indicated by: (a) a decrease in E6 and/or
E7 gene expression from high at the first time point to low at the
second time point; and (b) an increase in L1 gene expression from a
range of low to undetectable at the first time point to a range of
medium to high at the second time point.
37. The method of claim 13 wherein regression from a neoplastic to
a benign HPV infection is indicated by: (a) a decrease in E6 and/or
E7 gene expression from high at the first time point to low at the
second time point; (b) an increase in E2 gene expression from
undetectable at the first time point to high at the second time
point; and (c) an increase in L1 gene expression from a range of
low to undetectable at the first time point to a range of medium to
high at the second time point.
38. The method of claim 13 wherein progression from a benign to a
neoplastic HPV infection is indicated by: (a) an increase in E6
and/or E7 gene expression from low at the first time point to a
range of medium to high at the second time point; and (b) a
decrease in E2 gene expression from a range of low to high at the
first time point to a range of low to undetectable at the second
time point.
39. The method of claim 13 wherein progression from a benign to a
neoplastic HPV infection is indicated by: (a) an increase in E6
and/or E7 gene expression from low at the first time point to a
range of medium to high at the second time point; and (b) a
decrease in L1 gene expression from a range of medium to high at
the first time point to a range of low to undetectable at the
second time point.
40. The method of claim 13 wherein progression from a benign to a
neoplastic HPV infection is indicated by: (a) an increase in E6
and/or E7 gene expression from low at the first time point to a
range of medium to high at the second time point; (b) a decrease in
E2 gene expression from a range of low to high at the first time
point to a range of low to undetectable at the second time point;
and (c) a decrease in L1 gene expression from a range of medium to
high at the first time point to a range of low to undetectable at
the second time point.
41. The method of claim 13 wherein regression from a neoplastic to
a benign HPV infection is indicated by: (a) a decrease in E6 and/or
E7 gene expression from a range of medium to high at the first time
point to low at the second time point; and (b) an increase in E2
gene expression from a range of low to undetectable at the first
time point to a range of low to high at the second time point.
42. The method of claim 13 wherein regression from a neoplastic to
a benign HPV infection is indicated by: (a) a decrease in E6 and/or
E7 gene expression from a range of medium to high at the first time
point to low at the second time point; and (b) an increase in L1
gene expression from a range of low to undetectable at the first
time point to a range of medium to high at the second time
point.
43. The method of claim 13 wherein regression from a neoplastic to
a benign HPV infection is indicated by: (a) a decrease in E6 and/or
E7 gene expression from a range of medium to high at the first time
point to low at the second time point; (b) an increase in E2 gene
expression from a range of low to undetectable at the first time
point to a range of low to high at the second time point; and (c)
an increase in L1 gene expression from a range of low to
undetectable at the first time point to a range of medium to high
at the second time point.
44. The method of claim 13 wherein progression from a benign to a
neoplastic HPV infection is indicate by: (a) an increase in E6
and/or E7 gene expression from a range of low to medium at the
first time point to a range of medium to high at the second time
point; and (b) a decrease in E2 gene expression from a range of low
to high at the first time point to a range of low to undetectable
at the second time point.
45. The method of claim 13 wherein progression from a benign to a
neoplastic HPV infection is indicate by: (a) an increase in E6
and/or E7 gene expression from a range of low to medium at the
first time point to a range of medium to high at the second time
point; and (b) a decrease in L1 gene expression from a range of
medium to high at the first time point to a range of low to
undetectable at the second time point.
46. The method of claim 13 wherein progression from a benign to a
neoplastic HPV infection is indicate by: (a) an increase in E6
and/or E7 gene expression from a range of low to medium at the
first time point to a range of medium to high at the second time
point; (b) a decrease in E2 gene expression from a range of low to
high at the first time point to a range of low to undetectable at
the second time point; and (c) a decrease in L1 gene expression
from a range of medium to high at the first time point to a range
of low to undetectable at the second time point.
47. The method of claim 13 wherein regression from a neoplastic to
a benign HPV infection is indicated by: (a) a decrease in E6 and/or
E7 gene expression from a range of medium to high at the first time
point to a range of low to medium at the second time point; and (b)
an increase in E2 gene expression from a range of low to
undetectable at the first time point to a range of low to high at
the second time point.
48. The method of claim 13 wherein regression from a neoplastic to
a benign HPV infection is indicated by: (a) a decrease in E6 and/or
E7 gene expression from a range of medium to high at the first time
point to a range of low to medium at the second time point; and (b)
an increase in L1 gene expression from a range of low to
undetectable at the first time point to a range of medium to high
at the second time point.
49. The method of claim 13 wherein regression from a neoplastic to
a benign HPV infection is indicated by: (a) a decrease in E6 and/or
E7 gene expression from a range of medium to high at the first time
point to a range of low to medium at the second time point; (b) an
increase in E2 gene expression from a range of low to undetectable
at the first time point to a range of low to high at the second
time point; and (c) an increase in L1 gene expression from a range
of low to undetectable at the first time point to a range of medium
to high at the second time point.
50. The method of claim 13 wherein progression from HPV-infected
normal tissue to CIN-I is indicated by: (a) an increase in L1 gene
expression from a range of low to undetectable at the first time
point to a range of medium to high at the second time point; and
(b) low to medium E6 and/or E7 gene expression at the second time
point.
51. The method of claim 13 wherein progression from CIN-I to CIN
II/II or cancer is indicated by: (a) a decrease in L1 gene
expression from a range of medium to high at the first time point
to a range of low to undetectable at the second time point; and (b)
a medium to high level of E6 and/or E7 gene expression at the
second time point.
Description
[0001] This application is a continuation application of U.S.
patent application Ser. No. 11/681,539, filed Mar. 2, 2007, which
is a continuation of Ser. No. 09/970,477, filed Oct. 4, 2001, which
is a continuation of Ser. No. 09/210,168, filed Dec. 11, 1998,
issued as Pat. No. 6,355,424 on Feb. 21, 2002, to which priority
under 35 U.S.C. .sctn.120 is claimed. This application also claims
benefit of U.S. patent application Ser. Nos. 60/082,167, filed Apr.
17, 1998, 60/070,486, filed Jan. 5, 1998, and 60/069,426, filed
Dec. 12, 1997.
FIELD OF THE INVENTION
[0002] The present invention is generally related to the field of
cytological and 10 molecular assays and specifically to the area of
assays for the assessment of disease using a sensitive assay for
diagnosis and prognosis of HPV-induced carcinoma.
BACKGROUND OF THE INVENTION
[0003] The detection and diagnosis of disease is of obvious
importance for the treatment of disease. Numerous characteristics
of diseases have been identified and many are used for the
diagnosis of disease. Many diseases are preceded by, and are
characterized by, changes in the state of the affected cells.
Changes can include the expression of viral genes in infected
cells, changes in the expression patterns of genes in affected
cells, and changes in cell morphology. The detection, diagnosis,
and monitoring of diseases can be aided by the assessment of such
cell states.
[0004] An aspect of the present invention relates to human
papilloma virus (HPV), which induces benign epithelial
proliferations of the skin and mucosa in humans and is associated
with anogenital neoplasias and carcinomas. The intact DNA of HPV is
supercoiled and thus resembles an endless loop of twisted telephone
handset cord. Inside this shell, the viral DNA is packaged in and
around proteins from the cell nucleus, histones, and associated
peptides, into a structure that resembles cellular chromatin.
(Turek, (1994)). Human papillomaviruses characterized to date are
associated with lesions confined to the epithelial layers of skin,
or oral, pharyngeal, respiratory, and, most importantly, anogenital
mucosae. Specific human papillomavirus types, including HPV 6 and
11, frequently cause benign mucosal lesions, whereas other types,
HPV 16, 18, and a host of other strains, are predominantly found in
high-grade lesions and cancer. All human and animal
papillomaviruses appear to share a similar genetic organization,
although there are differences in the functions of individual viral
genes and in their regulation. The most common genital HPV type
associated with cervical carcinoma, HPV 16, has been studied most
extensively.
[0005] All large open reading frames (ORFs) in HPV are on one DNA
strand. Papillomaviral mRNAs appear to be transcribed solely from a
single strand in infected cells. The viral genome can be divided
into three regions, the upstream regulatory region (URR), or long
control region (LCR), containing control sequences for HPV
replication and gene expression, the viral early gene region,
encoding, among others, the E2, E6 and E7 genes, and the late
region, encoding the L1 and L2 genes. (Turek, (1994)).
[0006] HPV gene expression in high-grade premalignant disease or
cancer appears restricted to the early genes, possibly due to
cellular differentiation arrest induced by the viral E6 and E7
genes. In comparison to active HPV infection, E6 and E7 gene
control in cancer is deranged by mutations in the viral URR and, in
integrated viral fragments, by the disruption of the viral E2 gene,
stabilization of E6 and E7 mRNAs, and influences at the cellular
integration site.
[0007] Because the E2 gene is disrupted or inactivated in
integrated HPV fragments in invasive cervical carcinomas (Cullen,
(1991); Diirst, (1985); Matsukura, (1989); Schneider-Gadicke,
(1986); Schwarz, (1985); Wilczynski, (1988)), it has been predicted
that loss of E2 bestows a selective growth advantage to the
infected cell because of uncontrolled E6 and E7 expression
(Schneider-Gadicke, (1986); Schwarz, (1985)). Indeed, cervical
cells containing replicating HPV genomes rapidly segregate and are
outgrown in culture by cells that contain integrated viral genomes
(Jeon (1995)), but the underlying mechanism(s) have remained
unclear until recently. The full-length HPV 16 E2 gene products are
strong transcriptional activators comparable to HPV 1 E2 at some
viral as well as at simple, synthetic promoters (Demeret (1994);
Ushikai (1994)).
[0008] Genes E6 and E7 are considered to have oncogenic activity.
The encoded proteins interact with and disturb the physiologic
functions of cellular proteins that are involved in cell cycle
control. The E6/E7 proteins of HPV 16, 18 or related types are most
efficient in this regard. Some of these activities lead to genetic
instability of the persistently infected human cell. This enhances
the probability of mutations in cellular proto-oncogenes and tumor
suppressor genes and thus contributes to tumor progression.
Mutations in cellular genes devoted to the intracellular
surveillance of HPV infections, integration of viral DNA, and
deletions or mutations of viral transcription control sequences
lead to a significantly increased expression of the E6/E7 genes,
which is a consistent characteristic of high-grade intraepithelial
neoplasia and cancers. The genetic instability caused by viral
oncoproteins and the autocatalytic increase in oncoprotein
expression caused by mutations in the viral and cellular genome
identify the virus as a major driving force of progression to
carcinoma.
[0009] Individual types of human papillomaviruses (HPV) which
infect mucosal surfaces have been implicated as the causative
agents for carcinomas of the cervix, anus, penis, larynx and the
buccal cavity, occasional periungal carcinomas, as well as benign
anogenital warts. The identification of particular HPV types is
used for identifying patients with premalignant lesions who are at
risk of progression to malignancy. Although visible anogenital
lesions are present in some persons infected with human
papillomavirus, the majority of individuals with HPV genital tract
infection do not have clinically apparent disease, but analysis of
cytomorphological traits present in cervical smears can be used to
detect HPV infection. Conventional viral detection assays,
including serologic assays and growth in cell culture, are not
commercially available and/or are not suitable for the diagnosis
and tracking of HPV infection. Papanicolaou tests are a valuable
screening tool, but they miss a large proportion of HPV-infected
persons.
[0010] Thus, it is an object of the present invention to provide a
method for assessing the stage of HPV-based disease.
[0011] It is another object of the present invention to provide an
assay that can be combined with other assays to improve the
accuracy and reliability of prognostic and diagnostic assessments
of HPV-based disease.
[0012] It is a further object of the present invention to provide a
method for assessing the risk that a patient infected with HPV will
develop HPV-based disease.
[0013] It is another object of the present invention to provide a
method for stratifying patients who are currently HPV-infected but
without detectable HPV-based disease into those at risk for
progression to disease and those not at risk for progression to
disease.
[0014] It is also an object of the present invention to provide a
method for identifying treatment regimes for patients having
HPV-based disease.
[0015] Yet another object of the present invention to provide a
method for monitoring the effectiveness of treatment of HPV-based
disease.
[0016] A further object of the present invention to provide kits
for assessing the stage of HPV-based disease.
[0017] Another object of the present invention to provide
computer-based operation, analysis, and data management of assay
data to assess the stage of HPV-based disease.
SUMMARY OF THE INVENTION
[0018] One embodiment of the present invention involves measuring
the levels of expression of genes involved in a cell state, and
comparing their expression to each other or to reference genes in a
specific ratio, as an indication of the state of a disease in the
cell sample. The present invention can be used to assess the stage
or risk of a disease as indicated by the state of the cells. It can
also be used to guide or assess the effectiveness of a therapy for
a disease by identifying appropriate therapy based on the indicated
cell state or by indicating any change in the state of cells
subjected to the therapy.
[0019] In one form of the present invention, the stage and
prognosis of a human papillomavirus (HPV) infection or HPV-based
disease is assessed. This embodiment involves the measurement of
the level of expression of two or more HPV genes. Genes for this
purpose are the HPV E6, E7, L1, and E2 genes, although other HPV
genes such as E1, E4, E5, and L2 can also be used. It has been
discovered that the level of expression of these genes, the ratio
of expression of these genes to each other or to reference genes,
or both, are indicative of the stage of HPV-based disease.
[0020] Gene expression levels are used according to this invention
to assess the progression of HPV infection from benign to malignant
growth. HPV infection progresses from CIN I through CIN TTT and
finally to malignant cancer. These stages can be identified by the
ratios of HPV genes. In particular, the transition from CIN I to
CIN II/III, i.e., a transition to pre-malignancy, can be predicted
when the ratio of the present invention exceeds one. A ratio of
greater than 3 in the present invention indicates a transition from
pre-malignancy to malignant cancer. Thus, a ratio below 1 indicates
a low-level of CIN in an HPV infected cell. A ratio between one and
about three indicates a high grade CIN (i.e., CIN III) or
pre-malignant condition. And a ratio of over about three is an
indication of an HPV induced malignancy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a graph of detection of E6/E7 mRNA shown as the
number of cells/well versus the number of E6/E7 mRNA/well.
[0022] FIG. 2 is a graph of the sensitivity of an HPV L1 assay
shown as the number of RNA molecules/well versus the
signal-to-noise ratio minus 1.
[0023] FIG. 3 is the probe sequence for HPV 16 L2 (SEQ ID NO:
2).
[0024] FIG. 4 is the probe sequence for HPV 16 L1 (SEQ ID NO:
2).
[0025] FIG. 5 is the probe sequence for HPV 16 E6/E7 (SEQ ID NO:
3).
[0026] FIG. 6 is the probe sequence for HPV 16 E2 (SEQ ID NO:
4).
[0027] FIG. 7 is the probe sequence for HPV 16 E4 (SEQ ID NO:
5).
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention relates to the identification and
monitoring of diseased cells. One method involves measuring the
levels of expression of genes involved in a disease state, and
comparing their expression to each other or to reference genes, as
an indication of the state of the cells. Such measurements can be
combined with other assays to increase the accuracy and reliability
of the assessment of the disease state. One method of the present
invention can be used to assess the stage of a disease as indicated
by the state of the cells. This method can also be used to guide or
assess the effectiveness of a therapy for a disease by identifying
appropriate therapy based on the indicated disease state or by
indicating any change in the state of cells subjected to the
therapy.
[0029] Many diseases are characterized by specific cellular
phenotypes and gene expression patterns. For example, neoplastic
and cancerous cells generally exhibit certain distinctive
morphologies and growth characteristics. Molecular characteristics,
such as gene mutations and gene expression patterns are also a good
indicator of disease progression. Virally infected cells can
exhibit different morphologies and gene expression patterns,
including expression of viral genes. Using the present invention,
the characteristics of the cell state, such as changes in cell
morphology or expression of genes can be determined from a patient
sample.
[0030] The characteristics to be detected are generally specific to
the cell state of interest and the disease suspected of being
present in the cell sample. Such characteristics can be generally
divided into two types, cytological characteristics and molecular
characteristics. As used herein, cytological characteristics are
characteristics such as, for example, overall cell shape and
appearance. The primary identification and classification of many
neoplastic and cancerous cells has traditionally been accomplished
using cytological characteristics. Identification of cytological
characteristics is generally slow, requires a relatively high level
of training, and generally cannot be easily automated. As used
herein, molecular characteristics are the presence and state of
particular molecular species, such as proteins, nucleic acids, and
metabolites. Such molecular characteristics are generally
identified by detecting and measuring the particular molecules of
interest.
[0031] The characteristics assayed can include additional or
surrogate marker characteristics that are not a direct cause or
result of the disease but that are related to certain disease and
cell states. Examples of such additional markers include
polymorphic markers, human leukocyte antigens (HLA) such as B7 that
predispose women for cervical carcinomas, oncogenes, p53 mutations,
other cancer markers, oncosupressors, cytokines, growth factor
receptors, and hormones. Such markers can be present in, or absent
from, cells exhibiting state-or disease-specific characteristics,
and such presence or absence can be indicative of for example, a
more severe or less severe disease state. These markers can be used
in conjunction with the disclosed method to infer either higher or
lower risk of neoplastic disease depending on the number of
abnormal scores or the magnitude of change in quantitative
markers.
[0032] Examples of disease states for assessment using the present
invention include, but are not limited to neoplasias and cancer.
Disease states of interest are HPV-based disease--including HPV
infection, cervical intraepithelial neoplasia (CIN), and cancer,
atypical squamous cells of undetermined significance (ASCUS),
warts, condylomata, epidemic) dysplasia verruciformis and other
skin diseases, laryngeal papilloma, oral papilloma and conjunctival
papilloma.
[0033] An embodiment of the present invention is detection and
measurement of the expression levels of certain HPV genes. An
impressive amount of data has been accumulated over the past
decade, showing that carcinoma of the cervix is associated with
infection of certain types of HPV. Though the presence of HPV DNA
in a precancerous lesion is indicative of an increased relative
risk for cervical dysplasia and invasive carcinoma, it is still
difficult to predict the clinical behavior of precancerous cervical
lesions. Tumors arise due to the accumulation of genetic
alterations which can activate oncogenes and/or inactivate tumor
suppressor genes and/or genes involved in DNA damage recognition
and repair.
[0034] The expression levels of E6 and E7 oncoproteins encoded by
high-risk HPV types are a more sensitive and accurate measure of
potential risk of an HPV infection developing into a cancerous
lesion. The present invention measures the relative amounts of E6
and/or E7 expression levels and E2 and/or L1 in an HPV-infected
lesion to determine the ratio of E6 and/or E7 to L1 and/or E2,
where in this ratio is a direct measure of risk, and susceptibility
to the development of a cancerous lesion. HPV expression can be
measured by mRNA or protein levels in the cell.
[0035] In one aspect of the invention, the stage and prognosis of a
human papillomavirus (HPV) infection or HPV-based disease is
assessed. This embodiment of the present invention involves the
measurement of the level of expression of one or more HPV genes
discovered to be related to the stage and nature of HPV-based
disease. Genes 25 useful for this purpose include the HPV E6, E7,
L1, and E2 genes. It has been discovered that the level of
expression of these genes, the ratio of expression of these genes
to each other or to one or more other genes, or both, are
indicative of the stage of HPV-based disease. The level of
expression is relative to other HPV genes, or the level of
expression relative to a non-HPV gene, referred to herein as a
reference gene. Such reference genes can be any appropriate gene
(not encoded by HPV), and are, for example, housekeeping genes or
other constitutively expressed genes. Examples of reference genes
include actin genes, cytoskeletal genes, histone genes, tubulin
genes, epidermal growth factor receptor genes, the normal p53 gene,
the normal pRB gene, cyclin genes, 13-globin genes, and
glucose-6-phosphate dehydrogenase genes. Expression of reference
genes can be measured in the same cell as the level of HPV genes
are measured or in neighboring cells in the same cell sample. In
such a case, the reference gene is an internal control for gene
expression.
[0036] The relationship of the relative level of expression of
these genes to the state of cells infected with HPV is generally
illustrated in Table 1 below.
TABLE-US-00001 TABLE 1 Medical HPV-Based disease E6, E7, Character
state E6 + E7 E2 Ll benign HPV-infected low low to high low to
normal tissue undetectable benign Low grade ClN low to low to high
medium to i.e., CIN I medium high neoplastic High grade CIN medium
to low to low to i.e., CIN HMI high undetectable undetectable
neoplastic Cancer medium to low to low to high undetectable
undetectable
[0037] The HPV-based high grade CIN referenced in the table is the
premalignant stage leading to cancer and low grade CIN is a
productive viral infection that has little malignant potential but
is a public health concern with respect to the spread of HPV
infection. Normal tissue refers to cytologically normal tissue that
is infected with HPV. Although not limited to this standard, one
standard for establishing this lower limit of expression is a level
below that detectable using the hybrid capture assay described in
WO 93/10263 by Digene. As used herein, E6/E7 refers to E6, E7, or
E6 +E7. The addition of genes, as with "E6 +E7," for example,
refers to the combined expression of the added genes.
[0038] As can be readily discerned, each major disease state is
represented by a unique expression pattern of these three sets of
genes. Both of these conditions are regarded as serious medical
aliments. Other relationships involving the relative level 15
expression of other HPV genes (such as E1, E4, E5, and L2), and
other, non-HPV genes, can also be used to assess cell state. For
example, L2 and E4 are frequently associated with benign viral
production diseases, and E1 is similar in profile to E2 and is
often deleted in malignancies. Other relationships of expression
for these HPV proteins can exist for other HPV-based diseases, and
the disclosed method can be used to assess the state of such other
diseases using the appropriate levels and ratios for that
disease.
[0039] Using information about the levels and ratios of HPV genes
in different cell states, the stage of the disease can be assessed
in several ways. In some cases, the presence or absence of
detectable expression is indicative of the disease state in the
infected cells. For example, a lack of E2 expression (when HPV is
present) is indicative of high grade CIN or cancer. In other cases,
a change or difference in expression of an HPV gene product can be
indicative of change occurring in the infected cell state. For
example, an increased level of expression of E6 and E7--relative
to, for example, an earlier sample or a reference sample--may be
indicative of high grade CIN or cancer. A change in the ratio of
E6/E7 expression to E2 expression is used to identify low grade CIN
or a shift from normal cells to low grade CIN. Many other
combinations of comparisons are also possible, and other
combinations can be derived from the information in Table 1 for use
in the method of the present invention.
[0040] One way in which ratios of HPV genes can be related to
HPV-based disease states is by reference to groups of HPV genes.
For this purpose, group 1 genes or gene sets include E6, E7, and
E6+E7. Group 2 genes or gene sets include L1, L2, E4, and any
combinations. Group 3 genes or gene sets include E1, E2, E5, and
any combinations. Useful ratios of expression include ratios of
members of group 1 to members of group 2 or group 3. Examples of
theses ratios are (E6+E7)/(L1+L2), E6/L1, E7/L1, E6/L2, E7/L2,
(E6+E7)/L1, (E6+E7)/L2), E6/(L1+L2), and E7/(L1+L2). For such
ratios, a value of less than two is indicative of benign human
papillomavirus infection or low grade intra-epithelial neoplasia.
This type of infection is also classified as CIN I. HPV infections
which progress beyond CIN I indicate cell transformation has
occurred and cancerous growth has begun. These later infections
(CIN FM) have ratios of greater than 2. A ratio of expression of
more than two is indicative of high grade intra-epithelial
neoplasia or pre-malignant cancer. A ratio of expression of much
more than two (i.e., exceeding 4 and up to infinity) is indicative
of cancer. Preferred ratios for use in this invention are
(E6+E7)/L1, (E6+E7)/(L1+L2), (E6+E7+E2+E4)/(L1+L2),
(E6+E7)/(E2+E4), (E6+E7)/E2, (E6+E7+E2-E4)/(L1+L2).
[0041] There are several ways in which measured levels of
expression of HPV genes can be compared and categorized. For
example, where the presence or absence of expression is indicative
of the cell state, expression of the HPV gene is analyzed without
reference to the expression level of other genes. Where the
relative level of expression of an HPV gene is indicative of the
cell state, the measured level of expression is compared, for
example, to the level of expression of the same type of HPV gene in
a different cell sample (such as an earlier cell sample from the
same source or reference cells harboring HPV), to the level of
expression of a different type of HPV gene in the same or a
different cell sample, to the level of expression of a non-HPV
reference gene in the same cell sample, or to the level of
expression of a non-HPV reference gene in reference cells.
[0042] In one embodiment of the present invention, levels and
ratios of expression of HPV genes are compared to the levels of the
same genes in a cell line that contains HPV (such as HeLa or
CaSki). Such cell lines provide a standard against which levels of
expression of HPV genes in cell samples are compared. Such
comparisons are used to assess and compare the absolute levels of
expression of these HPV proteins with those in a standard or
comparative cell line. Other cell lines useful for this purpose are
non-cancerous cell lines infected with HPV 16 (such as W12) or HPV
31 (such as ON612; Meyers (1992)).
[0043] The levels and ratios of expression of HPV genes are also
compared to reference genes, such as housekeeping genes or other
constitutively expressed genes, in the same cells or in reference
cells, such as a cell line. For example, the level of expression of
the HPV gene and the reference gene is measured in the same cell
sample. Such measurements provide an internal control of the
overall expression level in a cell sample and are used to calculate
a corrected level of expression for the HPV gene to allow more
accurate comparisons of the level of expression between different
cell samples. One form of correction is referred to as
normalization. Thus, the level of expression of one or more HPV
genes can be measured in two or more cell samples along with the
level of expression of the same reference gene in each of the cell
samples. The level of expression of the HPV genes is then
normalized to each other based on differences, if any, between the
measured level of expression of the reference gene in each of the
samples.
[0044] In some stages of HPV-based disease, the expression of a
particular HPV gene is low or undetectable. Such lack of detectable
expression is used herein to identify patterns of expression that
serve as prognostic or diagnostic indicators. The expression of
other HPV genes is assessed in parallel or in the same assay to
provide a control for the lack of expression of one or more of the
genes to be assessed. This is accomplished by measuring the
expression of the several HPV genes together or in parallel. For
example, measuring the level of expression of the HPV E6, E7, L1,
E4, and E2 genes in parallel is useful since at least one of these
genes is expressed in all of the stages of HPV-based disease. In a
preferred embodiment of the present invention, a ratio of E6/E7 to
L1 /E2 provides an indicator of likelihood of developing cancer
from the HPV infection. In this way, indicative information is
collected as well as providing an internal control. The presence of
L1, L2, and E4 in combination is also indicative of benign disease
while the absence of E1 and/or E2 is indicative of neoplastic
potential.
[0045] The types of comparison described above can also be used
with other genes and other disease states. That is, the measured
level of expression of a gene of interest can be compared, for
example, to the level of expression of the same type of gene in a
different cell sample (such as an earlier cell sample from the same
source or appropriate reference cells), to the level of expression
of a different type of gene in the same or a different cell sample,
to the level of expression of a reference gene in the same cell
sample, or to the level of expression of a reference gene in
reference cells.
[0046] Expression of genes of interest, such as the HPV E6, E7, L1,
E4, and E2 genes, can be assessed using any suitable method. For
example, RNA can be detected using hybridization, amplification, or
sequencing techniques, and protein can be detected using specific
antibodies. Many techniques for the specific detection of gene
expression, by detection of expression products, are known and can
be used with the disclosed method. One technique for detecting and
measuring the level of expression of genes of interest is detection
of RNA transcribed from the genes of interest. For the most
reliable comparisons, expression levels that are to be compared
should be measured using the same technique and be performed in the
same manner.
[0047] For hybridization detection of HPV nucleic acids, a mixture
of probes specific for these sequences from different HPV types can
be used. This ensures that the method will detect expression
regardless of the type of HPV involved. For some purposes, it may
be desirable to use probes designed for the sequence of a certain
HPV type, or a mixture of probes for only some HPV types. Such
probes may or may not be type-specific depending on the differences
between the sequences of the HPV nucleic acids to be detected. One
useful mixture for this purpose would include probes for HPV types
more closely associated with a progression to cancer. The HPV types
most commonly associated with cervical cancer are types 16 and
18.
[0048] Useful techniques for measuring the level of expression of a
gene of interest in a cell sample include the hybrid capture
technique described in WO 93/10263 by Digene, PCR in situ
hybridization techniques described by (Nuovo, 1997)), branched DNA
assays (Chernoff (1997)), transcription-mediated amplification
(TMA); Stoflet (1988)), and polymerase chain reaction (PCR), ligase
chain reaction (LCR), self-sustained sequence replication (3SR),
nucleic acid sequence based amplification (NASBA), strand
displacement amplification (SDA), and amplification with QB
replicase (Birkenmeyer and Mushahwar, (1991); Landegren,
(1993)).
[0049] Numerous assays for the detection and measurement of gene
expression products are known and can be adapted for the
determination of the level of expression of genes of interest in
the disclosed assay. For example, many of the techniques for the
detection of HPV in general or expression of other HPV genes
described below can also be adapted for use in the disclosed assay
for the detection of expression of HPV genes E6, E7, L1, E4,and
E2.
[0050] Many HPV detection and typing assays, which can be used in
the disclosed method, are known, including assays involving
Southern blots, dot blots, in situ hybridization, polymerase chain
reaction, and solution hybridization. Mant (1997) describes PCR
assays used to identify DNA from specific HPV types. Cope (1997)
describes a PCR-based test using a consensus primer and a Hybrid
Capture assay (HCA) of detection of HPV types. The hybrid capture
assay is also described in WO 93/10263 by Digene. The hybrid
capture assay is a useful method for detection of HPV and for
determining HPV type in combination with the disclosed assay.
[0051] Swan (1997), discloses an HPV detection assay exploiting the
5' to 3' exonucleolytic activity of Taq DNA polymerase to increase
the signal from fluorescent dyes by releasing them from
genotype-specific probes during PCR. Lizardi (1997), describes a
method of detecting HPV using in situ hybridization with
non-radioactive probes and visualization with conventional
bright-field or fluorescence microscopy, or laser scanning confocal
microscopy. Zehbe [1] (1997), describes a modified version of in
situ hybridization for detection of HPV that involves signal
amplification. Leiserowitz (1997), describes use of reverse
transcriptase-polymerase chain reaction to detect HPV. Zehbe [2]
(1997), describes a nonisotopic, enzyme-linked immunosorbent
assay-based sandwich capture hybridization assay for HPV
detection.
[0052] In one embodiment of the present invention RNA was analyzed
directly by solution based procedures. The cells were first lysed
by adding a proteolytic enzyme to the cells contained in wells of a
microtiter plate. Non-limiting examples of enzymes for use in the
present invention include proteinase K or Pronase. Cells can also
be subjected to detergent lysis or osmotic lysis or a French Press.
After incubation, biotinylated DNA probes were added to each well.
The RNA:DNA hybrids were captured onto a solid phase by
transferring to streptavidin coated microplates. Alkaline
phosphataseconjugated antibodies to RNA:DNA hybrids were added to
each well in the hybridization microplate and signals were
generated by adding a chemiluminescent reagent such as
CDP-Star.RTM. with Emerald II (Tropix) to each well. The signal was
read from the microplate. The solution based DNA analysis was
performed similarly to the RNA analysis except that the microtiter
plates were coated with anti-RNA:DNA hybrid antibodies and the
probes were RNA probes.
[0053] Other methods for detection and assessment of HPV infections
that can be used in the disclosed method are described in U.S. Pat.
Nos. 5,415,995, 4,777,239, 5,484,699, 4,983,728, 5,527,898,
5,364,758, 5,639,871, 5,501,947, 5,665,533, 4,748,109, 5,623,932,
5,665,571 and 5,648,459. These are just examples of HPV detection
and typing assays that may be used in combination with the
disclosed molecular assay. Many of the techniques described above
can also be adapted for use in the disclosed assay for the
detection of expression of HPV genes.
[0054] The disclosed assay and other assays can also be
sequentially combined. That is, first one type of assay can be
performed, and then, depending on the results, another assay can be
performed. For example, an assay to detect HPV (or HPV type) can be
performed first, then, if HPV is present, the disclosed molecular
assay can be 25 performed. As another example, the disclosed
molecular assay can be performed first, and if the results of the
assay were indicative of a high grade CIN or cancer, a cytological
assay or biopsy can be performed. Such sequential combinations are
particularly useful for limiting more extensive testing to patients
and samples that are identified as high risk.
[0055] Useful sequential orders for the assays are (1) an HPV
assay, followed by an assay for one or more other markers, followed
by a cytological or histological assay; (2) a cytological or
histological assay, followed by an HPV assay or an assay for one or
more other markers, followed by an HPV assay or an assay for one or
more other markers (whichever had not been performed first); (3) a
cytological or histological assay, followed by a combined or
simultaneous HPV and marker assay; (4) a combined or simultaneous
HPV and marker assay, followed by a cytological or histological
assay; and (5) detection of HPV, detection of HPV type, an HPV
assay, and a cytological or histological assay. Each combination of
assays is followed by an assessment, using the combined assay
results, of the cell state, disease state, patient status, patient
prognosis, or other assessment as described herein.
[0056] Where the results of initial assays are either equivocal or
suggest a more severe stage of disease, further assays are useful
to clarify and confirm the initial results. For example, where a
cell sample is a mosaic with some cells benignly infected with HPV
and others exhibiting high grade neoplasia or cancer, an assay
measuring expression of HPV genes may give equivocal results. By
following up with a morphological assay, the presence of the high
grade neoplastic or cancerous cells can be established. In this
case, the benefit of the disclosed method is that only some of the
cell samples assayed (those with either equivocal or severe
results) need be tested further.
[0057] The disclosed method can also be combined with treatment
regimes. For example, results in the disclosed assay or method
indicative of high grade CIN or cancer suggest that antiviral
therapy will be ineffective since these stages of disease are often
25 accompanied by integration of HPV into the genome. On the other
hand, assay or method results indicative of normal or low grade
stages of disease suggest antiviral treatments since HPV is
generally not integrated at these stages. Antiviral treatments
include, for example, drugs or therapeutic vaccines. Where results
of the disclosed method indicate a benign cell state, treatment can
be avoided altogether. The ability to make such assessments
reliably and accurately is a significant benefit of the disclosed
assay.
[0058] The disclosed method can include the combination of a
molecular assay as described above with any other assay for
assessing a disease or state of cells in a cell sample. For
example, a molecular assay measuring the level or ratio of
expression of HPV genes can be combined with cytological assays,
histological assays, determination of the HPV type present,
determination of the level of HPV present, assays detecting other
cellular markers such as oncoproteins or tumor suppressors, or
combinations of these assays. Such assays are known and are used
for the diagnosis of HPV infection or HPV-based disease and
assessment of the stage of disease. Results from a molecular assay
and one or more additional assays can be combined to increase the
reliability of any assessment, prognosis, diagnosis, or monitoring
of HPV-based disease. This possibility of combination is a
particularly useful aspect of the disclosed method since the HPV
molecular assays described above provide information about
HPV-based disease that is distinct from other assays. Where
multiple assays point in the same prognostic or diagnostic
direction, the reliability of the assessment is increased. Useful
combinations include a cytological assay and the disclosed
molecular assay, an assay for determining HPV type and the
disclosed molecular assay, and a combination of a cytological
assay, a typing assay, an assay for detecting cancer markers, and
the disclosed molecular assay. Combined assays can be performed in
any order and in any temporal relationship. For example, various
assays can be performed in parallel or simultaneously. Such assays
can be performed in any manner such as on the same apparatus by the
same person, with different apparatus, or in the same or different
locations.
[0059] Cytological assays for use in assessing the stage of
HPV-based disease are known and can be used in the disclosed
method. The well established Pap smear and Hematoxylin & Eosin
stains (H&E) are preferred examples. The use and analysis of
Pap smears and H&E stains are well-known in the art.
[0060] A cell sample as the term is used herein is primarily a
collection of cells from a patient. One method of obtaining cells
is through non-invasive means, which is defined herein as obtained
without the puncturing of a patient. Examples of non-invasive means
are, for example, cell samples obtained from urine or a nasal,
epithelial, cervical or other cell surface scrape. Patient cells
can also be obtained by other means including, for example, needle
biopsy or tissue biopsy.
[0061] The cell sample can be preserved in a collection medium
which allows for a combination of two or more assays of different
characteristics related to a cell state of interest. As used
herein, the assay or assays refer to detection or measurement of
specific characteristics, the results of which may be combined with
other such measurements of other characteristics to an overall
assessment of a cell suspected of being infected with one or more
diseases. These assays may include, for example, a combination of
morphological analysis and quantitation of a particular RNA or DNA
or protein whose levels provide a specific indication of the
presence or progression of a disease. Alternatively, for example,
the collection medium can be used to combine an assay identifying
the morphology of cells in a cell sample with one or more assays
identifying the HPV type involved, and, for example, identifying
whether the HPV type identified is a high risk or low risk HPV type
for the development of HPV-induced cell transformation and
cancer.
[0062] For example, sources of cell samples for assessing HPV-based
disease include cervix, vagina, vulva, anus, penis, larynx, buccal
cavity, lymph nodes, malignant deposits in any part of the human
body, and epidermis; all of which are known sites of HPV infection
and pathology.
[0063] Cell samples for use in the present invention can be
collected and stored in liquid medium. Examples of useful cell
collection media are STM (Digene), PreservCyt.RTM. (Cytyc), and
CytoRich.TM. (Autocyte). These media (PreservCyt.RTM. and
CytoRich.TM.) were developed for the collection of cytological
samples but can be adapted for use with molecular assays.
[0064] Cell samples for use in the method of the present invention
can be fixed or processed in any manner consistent with the assays
to be performed. For example, both cytological and molecular assays
can be performed using cells fixed on a solid substrate such as,
for example, a slide. The requirements of the assays to be
performed will generally identify the sample processing to be
used.
[0065] The present invention can be conveniently performed using
kits that include one or more of the materials needed for the
method, such as reagents and sample collection and handling
materials. For example, kits can include cell collection medium,
sample preserving reagents, reagents for specific detection of DNA
and/or expression products (RNA or proteins) of one or more of the
E2, E4, E5, E6, E7, L1 or L2 genes, and sample handling containers.
Useful reagents for detection of expression of the HPV genes are
nucleic acid probes for those genes. A kit may also contain control
samples or reagents, or reagents and materials for performing other
assays to be combined with the disclosed assay. In addition, the
kits can contain reagents for the separation of RNA and/or DNA from
other cellular components.
[0066] The present invention can be performed using devices adapted
to the method. Numerous devices for performing similar assays are
known and in use and can be adapted for use with the disclosed
assays and method. For example, devices are known for automating
all or a part of sample assays and sample handling in assays.
[0067] All or part of the disclosed method can be controlled or
managed using special purpose computer programs. The data collected
from the disclosed method, and data from any other assay used in
combination, can be compiled, analyzed, and output in various forms
and for various purposes using special purpose computer programs.
Such programs can be used with, or combined into, other patient or
data management computer programs. The usefulness of such a program
increases with the number of measurements or assessments to be
combined, and the relative importance of each type of measurement
to the overall assessment. Computer programs for use with the
disclosed method can be used on general purpose computers, or can
be incorporated into special purpose computers or computerized
devices for controlling the disclosed method, handling and
analyzing data from the disclosed method or both.
EXAMPLES
[0068] The examples herein are meant to exemplify the various
aspects of 10 carrying out the invention and are not intended to
limit the invention in any way.
Example 1
General Methods for Nucleic Acid Analysis
[0069] The assay for nucleic acids follows in general principle the
method for detecting HIV RNA by the Digene Hybrid Capture HW Test,
described in WO 93/10263 by Digene. Briefly, following lysis, 50
ill of probe mix (containing DNA biotinylated probe) was added to
each well. The plate was sealed and incubated at 65.degree. C. for
2 hours for hybridization to occur. After hybridization, samples
were transferred to a strepavidin-coated microplate, and 25 iaL of
anti-hybrid antibody was added to each well. The plate was agitated
at 1100 RPM, for 1 hour, at room temperature. Wells were washed
6.times. times with 65.degree. C. wash buffer, followed by one wash
using distilled water. 100 Ill of a chemiluminescent substrate was
added to each well and the plate was incubated at room temperature
for 30 minutes. The plate was then read in the DML 2000
luminometer. The data was then expressed as signal-to-noise. Using
a calibration curve, the chemiluminescent signal generated by each
specimen was converted into mRNA copies per cell. The assay
described above can be run on either whole lysed cells or nucleic
acid separated from other cellular components.
Example 2
Quantitation of HPV
[0070] This example illustrates the measurement of HPV E6/E7
expression for use in the disclosed method. A method for detecting
and quantitating HPV mRNA, including E6/E7 and mRNA has been
developed. This example measures expression in CaSki cells, but the
method is generally applicable to other cell lines and clinical
specimens. CaSki cells contain an integrated high-risk HPV-16
genome (about 600 copies/cell). CaSki cells were maintained in
subconfluence in RMPI 1640 media containing 10% FBS and 10 mM
sodium pyruvate. For this example, CaSki cells were grown to
confluence and were removed from the dishes by treatment with 0.1%
trypsin-0.5 mM EDTA. Using trypan blue, viable cells were counted
under microscopy. Cells were seeded, in 10 pl volumes, at final
concentrations of 10, 10.sup.2, 10.sup.3, 10.sup.4, and 105
cells/well in a polystyrene, tissue culture treated, 96 well plate.
Each dilution was performed and seeded in triplicate.
[0071] The cells were lysed with Proteinase K (30 units) in a
Tris-EDTA-buffered SDS). The plate solutions (20 mM Tris pH 7.4, 20
mM EDTA and 0.5' was sealed, agitated for 30 seconds at 1100 RPM
and incubated at 37.degree. C. for 30 minutes. The test for HPV
mRNA follows in general principle the method for detecting HIV RNA
by the Digene Hybrid Capture HIV Test, described in WO 93/10263 by
Digene. Briefly, following lysis, 50 pl of probe mix (containing
E6/E7 DNA biotinylated probe) was added to each well. The plate was
sealed and incubated at 65.degree. C. for 1.5 hours for
hybridization to occur. After hybridization, samples were
transferred to a strepavidin-coated microplate, and 25 of
anti-hybrid antibody was added to each well. The plate was agitated
at 1100 RPM, for 1 hour, at room temperature. Wells were washed
6.times. times with 65.degree. C. wash buffer, followed by one wash
using distilled water. 100 .mu.l of a chemiluminescent substrate
was added to each well and the plate was incubated at room
temperature for 30 minutes. The plate was then read in the DML 2000
luminometer. The data was then expressed as signal-to-noise. Using
a calibration curve, the chemiluminescent signal generated by each
specimen was converted into mRNA copies per cell. The data for the
direct detection of HPV mRNA from CaSki cells is shown in FIG. 1.
This method is exemplified by the detection and quantitation of
E6/E7 mRNA, but has also been applied to quantitate other mRNA
molecules (for example, HPV L1 mRNA) from CaSki cells (FIG. 2).
Example 3
Quantitation of HPV mRNA Using Preservative Collection Medium
[0072] CaSki cell line was trypsinized by incubating with 0.25%
Trypsin-EDTA for 5 minutes at 37.degree. C. Cells were pelleted
from the suspension by centrifugation at 800 rpm for 3 minutes in
Sorvall RT 6000 centrifuge. Cell pellet was resuspended in 500
.mu.l of 1.times. PBS and counted under microscope Trypan Blue
solution. Cells were diluted to 50 and 500 cells/.mu.l in 1.times.
PBS. 10 .mu.l of each cell concentration, including zero point (10
.mu.l of 1.times. PBS) were spiked in 3 ml of PreservCyt reagent.
100 .mu.l of Sample Conversion Buffer were added into each tub to
help visualize the cell pellet. All samples were mixed well and
were spun down at 3800 rpm for 15 minutes in Sorvall RT 6000
centrifuge. Supernatants were discarded and tubes were drained by
inversion on the Kimtowels for 2 to 5 minutes on the bench. All
pellets were resuspended with 50 .mu.l of the lysis reagent (50
units of Proteinase K) and mixture was transferred into the plate
coated with streptavidin. Plate was covered with the plate sealer
and was incubated at 37.degree. C. for 30 minutes (heat block) with
agitation every 15 minutes.
[0073] 50 .mu.l of each E6/E7 RNA calibration were loaded in
designated wells at 0, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6,
10.sup.7 and 10.sup.8 molecules/well to construct calibration curve
for mRNA in the specimen. 50 .mu.l of the probe mix was added into
each well. Plate was covered with the plate sealer and was agitated
for 1 minute at 1100 rpm on the bench top shaker. Samples were
incubated at 65.degree. C. for 1.5 hours (hybridization reaction)
in the heat block. Plate was transferred into the bench top shaker
and was incubated for 1 hour with agitation at 1100 rpm at room
temperature (capture reaction). 25 .mu.l of Detection Reagent 1 was
added into each well and plate was incubated without agitation for
1 hour at room temperature.
[0074] The contents of the plate were discarded and the plate was
washed vigorously six times with Wash Buffer at 65.degree. C. and
one time with deionized water at room temperature. The plate was
drained into Kimtowels and 100 .mu.l of the Detection Reagent 2 was
added into each well. Plate was incubated for 30 minutes at room
temperature covered from the light. At the end of incubation time,
plate was read on the Digene DML 2000 luminometer and the data were
expressed as signal-to-noise.
Example 4
Comparison of the Expression of E6/E7 RNA and L1 RNA in Cell Lines
Containing Episomal and Integrated HPV 16 DNA
Cell Lines Tested
[0075] The following cell lines were examined according to the
procedures outlined above. [0076] HaCaT: an immortalized human
keratinocyte cell line (Boukamp (1988)) SiHa: a human cancer cell
line (Fried', (1970)) [0077] W12: a non-tumorigenic human cervical
keratinocyte cell line (Stanley, (1989))
HPV Infection Status
[0078] HaCaT cells were infected with HPV 16 by the procedure of
White et. al. (White (1998)) to produce an episomal
(non-integrated, total sequence, not spliced) HPV infection.
Approximately 1 copy of HPV16 was present for every 40 cells. These
cells are considered a representative of early stage infection or
CIN I (cervical intraepithelial neoplasia). W12 cells contain
approximately 100 copies of episomal HPV 16 DNA and represent
pre-malignant, immortalized cells or CIN II or CIN III. SiHa cells
contain 1-2 copies of HPV 16 integrated into the genome. These
cells are considered to represent cancer.
Procedure
[0079] The RNA analysis was done according to Example 1 or the
following procedure. Single stranded, biotinylated, DNA probes
containing the specific HPV16 gene sequences were prepared. For
HaCaT and SiHa cell lines, cells were grown to confluency, cells
were harvested, and the total RNA was purified using the RNeasy kit
(Qiagen Inc., Santa Clarita, Calif.). For W12, whole cells were
used for analysis. RNA calibrators containing the complete HPV
genome were prepared by transcribing (+) sense RNA from a plasmid
containing the complete HPV16 genome with T7 RNA polymerase. The
RNA was then diluted to 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, and
10.sup.7 copies per 50 pl. Aliquots of cellular RNA were diluted to
50 .mu.l and then 50 .mu.l of Probe mix (containing the
biotinylated, single-stranded DNA probe) was added and hybridized
to the RNA specimens for 2 hours at 65.degree. C. The hybridization
reactions were transferred to a streptavidin coated microplate and
25 .mu.l of Detection Reagent 1 was added to each well. (Detection
Reagent 1 contains the alkaline-phosphatase--anti-RNA:DNA
monoclonal antibody conjugate.) During a 1 hour incubation with
shaking, RNA:DNA hybrids were captured onto the streptavidin coated
plate and were simultaneously reacted with the antihybrid antibody
conjugate. After several wash steps, a chemiluminescent substrate
(Tropix CDP-star with Emerald) was added to the wells, and the
light output was measured in a microplate luminometer after 30
minutes incubation at room temperature.
Quantitation
[0080] The quantitation of HPV mRNA was performed as follows. The
results from the RNA calibrators were used to construct standard
curves. The regression equation was calculated from the logarithm
of the copies versus the logarithm of signal to noise minus one
[(S/N)-1)]. The regression equations were then used to calculate
the number of copies of mRNA in the cellular RNA samples.
Ratio Results
[0081] The ratios of HPV 16 E6, E7, E2, E4, L1 and L2 were
calculated for each cell type. The results are shown in Table 2.
These results demonstrate that in an episomal, early stage
infection the ratio of (E6+E7)/L1 is about 0.7, in the
pre-malignant immortalized cell line the ratio is about 4 and in
the cancerous cell line the ratio approaches infinity.
TABLE-US-00002 TABLE 2 HaCaT W12 SiHa HPV Status Episomal Episomal
Integrated Cell Status Early stage Pre- Malignant infection
malignant, immortalized (E6 + E7)/L1 0.68 4.00 .infin.* (E6 +
E7)/(L1 + L2) ND 3.47 59.9 (E7 + E6 + E2 + E4)/(L1 + L2) ND 6.96
70.6 (E6 + E7)/(E2 + E4) ND 1.00 5.60 (E6 + E7)/E2 ND 4.40 12.00
(E6 + E7 + E2 - E4)/(L1 + L2) ND 1.58 59.10 *L1 gene transcripts
were undetectable in SiHa cells. Therefore, ratios of other gene
transcripts to the Ll gene transcript are infinitely large.
[0082] Publications cited herein and the material for which they
are cited are specifically incorporated by reference.
[0083] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
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Sequence CWU 1
1
511390DNAHuman papillomavirusL2-HPV16 1ttgttgtata ccataactta
ctattttttc ttttttattt tcatatataa tttttttttt 60tgtttgtttg tttgtttttt
aataaactgt tattacttaa caatgcgaca caaacgttct 120gcaaaacgca
caaaacgtgc atcggctacc caactttata aaacatgcaa acaggcaggt
180acatgtccac ctgacattat acctaaggtt gaaggcaaaa ctattgctga
acaaatatta 240caatatggaa gtatgggtgt attttttggt gggttaggaa
ttggaacagg gtcgggtaca 300ggcggacgca ctgggtatat tccattggga
acaaggcctc ccacagctac agatacactt 360gctcctgtaa gacccccttt
aacagtagat cctgtgggcc cttctgatcc ttctatagtt 420tctttagtgg
aagaaactag ttttattgat gctggtgcac caacatctgt accttccatt
480cccccagatg tatcaggatt tagtattact acttcaactg ataccacacc
tgctatatta 540gatattaata atactgttac tactgttact acacataata
atcccacttt cactgaccca 600tctgtattgc agcctccaac acctgcagaa
actggagggc attttacact ttcatcatcc 660actattagta cacataatta
tgaagaaatt cctatggata catttattgt tagcacaaac 720cctaacacag
taactagtag cacacccata ccagggtctc gcccagtggc acgcctagga
780ttatatagtc gcacaacaca acaggttaaa gttgtagacc ctgcttttgt
aaccactccc 840actaaactta ttacatatga taatcctgca tatgaaggta
tagatgtgga taatacatta 900tatttttcta gtaatgataa tagtattaat
atagctccag atcctgactt tttggatata 960gttgctttac ataggccagc
attaacctct aggcgtactg gcattaggta cagtagaatt 1020ggtaataaac
aaacactacg tactcgtagt ggaaaatcta taggtgctaa ggtacattat
1080tattatgatt taagtactat tgatcctgca gaagaaatag aattacaaac
tataacacct 1140tctacatata ctaccacttc acatgcagcc tcacctactt
ctattaataa tggattatat 1200gatatttatg cagatgactt tattacagat
acttctacaa ccccggtacc atctgtaccc 1260tctacatctt tatcaggtta
tattcctgca aatacaacaa ttccttttgg tggtgcatac 1320aatattcctt
tagtatcagg tcctgatata cccattaata taactgacca agctccttca
1380ttaattccta 139021484DNAHuman papillomavirusL1-HPV16 2gaggccactg
tctacttgcc tcctgtccca gtatctaagg ttgtaagcac ggatgaatat 60gttgcacgca
caaacatata ttatcatgca ggaacatcca gactacttgc agttggacat
120ccctattttc ctattaaaaa acctaacaat aacaaaatat tagttcctaa
agtatcagga 180ttacaataca gggtatttag aatacattta cctgacccca
ataagtttgg ttttcctgac 240acctcatttt ataatccaga tacacagcgg
ctggtttggg cctgtgtagg tgttgaggta 300ggtcgtggtc agccattagg
tgtgggcatt agtggccatc ctttattaaa taaattggat 360gacacagaaa
atgctagtgc ttatgcagca aatgcaggtg tggataatag agaatgtata
420tctatggatt acaaacaaac acaattgtgt ttaattggtt gcaaaccacc
tataggggaa 480cactggggca aaggatcccc atgtaccaat gttgcagtaa
atccaggtga ttgtccacca 540ttagagttaa taaacacagt tattcaggat
ggtgatatgg ttcatactgg ctttggtgct 600atggacttta ctacattaca
ggctaacaaa agtgaagttc cactggatat ttgtacatct 660atttgcaaat
atccagatta tattaaaatg gtgtcagaac catatggcga cagcttattt
720ttttatttac gaagggaaca aatgtttgtt agacatttat ttaatagggc
tggtactgtt 780ggtgaaaatg taccagacga tttatacatt aaaggctctg
ggtctactgc aaatttagcc 840agttcaaatt attttcctac acctagtggt
tctatggtta cctctgatgc ccaaatattc 900aataaacctt attggttaca
acgagcacag ggccacaata atggcatttg ttggggtaac 960caactatttg
ttactgttgt tgatactaca cgcagtacaa atatgtcatt atgtgctgcc
1020atatctactt cagaaactac atataaaaat actaacttta aggagtacct
acgacatggg 1080gaggaatatg atttacagtt tatttttcaa ctgtgcaaaa
taaccttaac tgcagacgtt 1140atgacataca tacattctat gaattccact
attttggagg actggaattt tggtctacaa 1200cctcccccag gaggcacact
agaagatact tataggtttg taacccaggc aattgcttgt 1260caaaaacata
cacctccagc acctaaagaa gatgatcccc ttaaaaaata cactttttgg
1320gaagtaaatt taaaggaaaa gttttctgca gacctagatc agtttccttt
aggacgcaaa 1380tttttactac aagcaggatt gaaggccaaa ccaaaattta
cattaggaaa acgaaaagct 1440acacccacca cctcatctac ctctacaact
gctaaacgca aaaa 14843779DNAHuman papillomavirusE6/E7-HPV16
3acattttatg caccaaaaga gaactgcaat gtttcaggac ccacaggagc gacccagaaa
60gttaccacag ttatgcacag agctgcaaac aactatacat gatataatat tagaatgtgt
120gtactgcaag caacagttac tgcgacgtga ggtatatgac tttgcttttc
gggatttatg 180catagtatat agagatggga atccatatgc tgtatgtgat
aaatgtttaa agttttattc 240taaaattagt gagtatagac attattgtta
tagtttgtat ggaacaacat tagaacagca 300atacaacaaa ccgttgtgtg
atttgttaat taggtgtatt aactgtcaaa agccactgtg 360tcctgaagaa
aagcaaagac atctggacaa aaagcaaaga ttccataata taaggggtcg
420gtggaccggt cgatgtatgt cttgttgcag atcatcaaga acacgtagag
aaacccagct 480gtaatcatgc atggagatac acctacattg catgaatata
tgttagattt gcaaccagag 540acaactgatc tctactgtta tgagcaatta
aatgacagct cagaggagga ggatgaaata 600gatggtccag ctggacaagc
agaaccggac agagcccatt acaatattgt aaccttttgt 660tgcaagtgtg
actctacgct tcggttgtgc gtacaaagca cacacgtaga cattcgtact
720ttggaagacc tgttaatggg cacactagga attgtgtgcc ccatctgttc tcagaaacc
77941113DNAHuman papillomavirusE2- HPV16 4gaggacgagg acaaggaaaa
cgatggagac tctttgccaa cgtttaaatg tgtgtcagga 60caaaatacta acacattatg
aaaatgatag tacagaccta cgtgaccata tagactattg 120gaaacacatg
cgcctagaat gtgctattta ttacaaggcc agagaaatgg gatttaaaca
180tattaaccac caagtggtgc caacactggc tgtatcaaag aataaagcat
tacaagcaat 240tgaactgcaa ctaacgttag aaacaatata taactcacaa
tatagtaatg aaaagtggac 300attacaagac gttagccttg aagtgtattt
aactgcacca acaggatgta taaaaaaaca 360tggatataca gtggaagtgc
agtttgatgg agacatatgc aatacaatgc attatacaaa 420ctggacacat
atatatattt gtgaagaagc atcagtaact gtggtagagg gtcaagttga
480ctattatggt ttatattatg ttcatgaagg aatacgaaca tattttgtgc
agtttaaaga 540tgatgcagaa aaatatagta aaaataaagt atgggaagtt
catgcgggtg gtcaggtaat 600attatgtcct acatctgtgt ttagcagcaa
cgaagtatcc tctcctgaaa ttattaggca 660gcacttggcc aaccaccccg
ccgcgaccca taccaaagcc gtcgccttgg gcaccgaaga 720aacacagacg
actatccagc gaccaagatc agagccagac accggaaacc cctgccacac
780cactaagttg ttgcacagag actcagtgga cagtgctcca atcctcactg
catttaacag 840ctcacacaaa ggacggatta actgtaatag taacactaca
cccatagtac atttaaaagg 900tgatgctaat actttaaaat gtttaagata
tagatttaaa aagcattgta cattgtatac 960tgcagtgtcg tctacatggc
attggacagg acataatgta aaacataaaa gtgcaattgt 1020tacacttaca
tatgatagtg aatggcaacg tgaccaattt ttgtctcaag ttaaaatacc
1080aaaaactatt acagtgtcta ctggatttat gtc 11135262DNAHuman
papillomavirusE4-HPV16 5ctacatctgt gtttagcagc aacgaagtat cctctcctga
aattattagg cagcacttgg 60ccaaccaccc cgccgcgacc cataccaaag ccgtcgcctt
gggcaccgaa gaaacacaga 120cgactatcca gcgaccaaga tcagagccag
acaccggaaa cccctgccac accactaagt 180tgttgcacag agactcagtg
gacagtgctc caatcctcac tgcatttaac agctcacaca 240aaggacggat
taactgtaat ag 262
* * * * *