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Molecular diagnostic assays for cervical neoplasia emerging

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Molecular diagnostic assays for cervical neoplasia emerging Powered By Docstoc
					Molecular diagnostic assays for cervical
neoplasia: emerging markers for the
detection of high-grade cervical disease
Douglas P. Malinowski
TriPath Oncology, Durham, NC, USA
BioTechniques 38:S17-S23 (April 2005)

The accurate detection and diagnosis of cervical carcinoma and its malignant precursors (collectively referred to as high-grade cervical
disease) represents one of the current challenges in clinical medicine and cytopathology. The advent of molecular diagnostics and the use
of whole-genome profiling using DNA microarrays promises to yield improved understanding of the disease process with the subsequent
development of more accurate diagnostic procedures based upon these discoveries. Recent reports describing a variety of experimental
approaches have identified a series of candidate genes that are overexpressed in cervical carcinoma. In this article, representative examples
of these markers and the resulting translational research will be reviewed within the context of improved cervical disease detection. An
emerging class of markers, the minichromosome maintenance protein family of DNA licensing factors (MCM-2, MCM-6, MCM-7), shows
promise for the specific detection of high-grade cervical disease using simple antibody-based immunochemistry formats. These proteins are
overexpressed in cervical disease as a result of infection by oncogenic strains of human papillomavirus (HPV) and subsequent uncontrolled
activation of gene transcription and aberrant S-phase induction, mediated through the E2F transcription factor pathway. This behavior
appears to be a hallmark of high-grade cervical disease and provides the link between oncogenic HPV infections and the molecular behavior
of cervical neoplasia (CN). The use of these molecular descriptors of CN in simple immunochemistry formats compatible with conventional
cytology preparations is anticipated to improve the screening and detection of cervical disease within the healthcare system.


INTRODUCTION                                                                    The use of morphology-based classification for cervical neoplasia
    Cervical neoplasia (CN), which includes squamous cell                   presents a number of dilemmas for the accurate diagnosis of this
carcinoma arising from cells lining the exterior of the uterus cervix,      disease. Factors that compromise the diagnostic accuracy of Pap
adenocarcinoma arising from cells lining the endocervical canal,            screening and cytology diagnosis include rare event detection,
and their malignant high-grade dysplastic precursors, represent a           sample adequacy, obscuring features such as blood, mucous,
significant issue in women’s health on a world-wide basis. Each year        inflammation, and the appearance of suspicious appearing cells
in the U.S., approximately 14,000 new cases of cervical cancer and          that appear malignant yet are the result of benign cellular changes
approximately 500,000 cases of high-grade malignant precursors to           arising from inflammation, repair processes, etc. In addition, the
cervical cancer are detected in 50 million women who are screened           diagnosis of cervical disease based upon cellular morphology is
annually with a Papanicolaou (Pap) smear. Outside the U.S.,                 inherently a subjective analysis that is highly dependent upon the
cervical cancer represents a significant cause of mortality in women        skills and experience of the cytotechnologist and the cytopathologist,
with an estimated 470,000 new cases of cervical cancer diagnosed            which can lead to variability in diagnoses. Hence there is a
annually. The detection and diagnosis of CN is accomplished                 recognized need within the medical community to utilize
through the morphological assessment of cervical cells obtained             supplemental diagnostic markers to improve the sensitivity,
during an annual Pap screen examination. In this procedure,                 specificity, reproducibility, and utility of current morphology-based
scrapings of both endocervical and ectocervical cells from the cervix       diagnostics for the detection of high-grade cervical disease.
are collected, deposited on a microscope slide, stained with the
Pap stain, and then examined microscopically for the presence of
neoplastic cells as determined by abnormal morphology.
    Within the morphologic classification of CN, the malignant
precursors of squamous cervical carcinoma include severe and
moderate dysplasia that are defined respectively as cervical
intraepithelial neoplasia grade 3 (CIN3) and grade 2 (CIN2) in
histology and as high-grade squamous intraepithelial lesions (HSIL)
in cytology. Mild dysplasia is defined as CIN1 on histology and
low-grade squamous intraepithelial lesion (LSIL) on cytology and
generally represents the morphologic abnormality associated with
transient human papillomavirus (HPV) infections. However, under-
lying cervical disease can still be present within these LSIL or CIN1
specimens. Finally, suspicious cells that appear can be categorized
either as atypical squamous cells in which HSIL cannot be ruled
out (ASC-H) or atypical squamous cells of uncertain significance
(ASC-US). Appropriate clinical management guidelines exist to
manage patients across this spectrum of morphological classifica-           Figure 1. The presence of cervical highgrade squamous intraepithelial
tions of cervical abnormalities (1–3). For the purposes of this article,    lesion (HSIL) cells within a cervical cytology specimen. The HSIL cells are
                                                                            characterized by a smaller cell size and an enlarged nucleus in comparison to
high-grade cervical disease is defined as the detection of a CIN2           the larger normal cervical keratinocytes shown in the figure. Also shown in
lesion or higher following colposcopy examination and tissue biopsy         the figure are small inflammatory leukocytes. The cervical cytology specimen
confirmation. A typical high-grade HSIL cell within a cervical cytology     was collected in SurePath™ liquid-based fluid (Tripath Imaging, Burlington,
specimen is shown in Figure 1.                                              NC, USA).

April 2005                                                                                                          Biomarkers in Cancer Research     17
Table 1. Human Papillomavirus Open Reading Frame                             clinical trial helped define the clinical utility of HPV testing in
Genes                                                                        combination with annual Pap screening. Within the ASCUS patient
 HPV Gene         Function                                                   population, 50%–60% of these patients harbored HPV infections
                                                                             with 7% of patients presenting high-grade disease upon colposcopy
 LCR              Regulatory control of transcription, replication, and
                  host interactions.
                                                                             and biopsy confirmation. The ALTS trial concluded that HPV triage
                                                                             of ASCUS patients was more effective for disease detection upon
 L1               Major capsid protein.
                                                                             referral to colposcopy than repeat cytology or direct referral to
 L2               Minor capsid protein.                                      colposcopy. However, HPV testing of LSIL patients was not useful
 E1               Viral replication and maintenance of viral episome.        as a triage for colposcopy because of their high prevalence of HPV
 E2               Transcriptional regulation and cofactor for                infection (8–12). The use of HPV testing has been approved by
                  replication.                                               the Food and Drug Administration (FDA) in combination with Pap
 E4               Keratin interactions and viral shedding.                   screening as both a reflex test within the ASCUS patient population
 E5               Growth factor receptor interactions and signal             and as a primary screen for cervical disease (13). The utility for
                  transduction.                                              HPV testing in a primary diagnostic setting has been recommended
 E6 and E7        Prolongs division phase of the cell cycle to               in recent guidelines from the American College of Obstetrics and
                  promote replication.                                       Gynecology (ACOG) (14). In addition to the Hybrid Capture II assay,
                  Responsible for malignant transformation of the            a number of PCR-based methods have been shown to support HPV
                  cervical keratinocyte following infection with onco-       detection and viral genotyping using liquid-based cervical cytology
                  genic subtypes of HPV.                                     specimens (15–19).
 HPV, human papillomavirus.                                                      The clinical utility of HPV-based screening for cervical disease
                                                                             is in its negative predictive value. An HPV negative result in combi-
                                                                             nation with a history of normal Pap smears is an excellent indicator
HPV IN THE PATHOGENESIS OF                                                   of a disease-free condition and a low risk of CN during the subse-
CERVICAL NEOPLASIA                                                           quent 1–3 years. However, a positive HPV result is not diagnostic
                                                                             of cervical disease; rather it is an indication of infection. Although
     HPV is recognized as the etiologic agent responsible for the            the majority of HPV infections are transient and will spontaneously
initiation of CN. Within the HPV family of viruses, there are both non-      clear within a 12-month period, a persistent infection with a high-
oncogenic and oncogenic forms of the virus. The non-oncogenic                risk HPV viral subtype indicates a higher risk for the development
forms of the virus (including HPV types 6 and 11) are associated             of CN. To supplement HPV testing, a number of molecular markers
with common warts and condyloma. The oncogenic forms of HPV                  associated with CN have been evaluated in order to improve the
(including types 16 and 18) are associated with cervical carcinoma.          clinical specificity for cervical disease diagnosis.
The oncogenic forms of HPV can be classified into high-risk and
intermediate-risk viral subtypes. The high-risk HPV viral subtypes
include HPV types 16, 18, 45, and 58, and the intermediate-risk              HPV-INDUCED ALTERATIONS OF THE CELL
HPV viral subtypes include HPV types 31, 33, 35, 39, 51, 52, and 69          CYCLE AND CELLULAR PROLIFERATION
(4). The genome of HPV consists of a double-stranded circular DNA                HPV infection within cervical keratinocytes results in a number
molecule with eight open reading frames encoding the genes shown             of alterations that disrupt the activities within the cell cycle. The
in Table 1. Within the context of CN development, the genes E6 and           cell cycle normally represents a series of coordinated activities
E7 from the oncogenic forms of HPV encode two distinct oncopro-              within the cell that insures DNA integrity prior to replication of the
teins that are the major determinants in cellular transformation as          genome, the separation of duplicated chromosomes and final cell
discussed in more detail below.
     Current literature suggests that HPV infects
the basal stem cells within the underlying tissue
of the uterus cervix. Differentiation of the stem
cells into mature keratinocytes, with resulting
migration of the cells to the stratified cervical
epithelium, is associated with HPV viral replication
and reinfection of cells. During this viral replication
process, a number of cellular changes occur that
include cell cycle deregulation, active proliferation,
DNA replication, transcriptional activation, and
genomic instability (5–7). These changes are
diagramed in Figure 2.
     Most HPV infections are transient in nature, with
the viral infection resolving itself within a 12-month
period. For those individuals who develop persistent
infections with one or more oncogenic subtypes of
HPV, there is a risk for the development of neoplasia
in comparison to patients without an HPV infection.
Given the importance of HPV in the development
of CN, the clinical detection of HPV has become Figure 2. The pathogenesis of cervical neoplasia (CN). The general scheme for cervical cancer
an important diagnostic tool in the identification of pathogenesis begins with infection of the cervical epithelial stem cell with human papillomavi-
patients at risk for developing CN. Both the high- rus (HPV). Persistent infection of the differentiating keratinocyte results in a number of cellular
risk and the intermediate-risk HPV viral subtypes and genomic changes which include: (i) integration of the HPV virus into the host genome with
are detectable through the use of the DiGene linearization of the viral episome and deletion of the E2 gene; (ii) the overexpression of the on-
Hybrid Capture® II assay (DiGene Corporation, cogenes E6 and E7; (iii) the deregulation of the cell cycle with concomitant increase in cellular
Gaithersburg, MD, USA). The National Cancer proliferation;cells with enhanced growth characteristics and proliferative potential.clonal selection
                                                          of abnormal
                                                                      and finally (iv) the generation of genomic instability which leads to
Institute (NCI) ASCUS LSIL Triage Study (ALTS)
18   Biomarkers in Cancer Research                                                                                                          April 2005
division. Key proteins that coordinate these activities include the               G1 phase through direct interaction of E7 with the Smad proteins
tumor suppressor protein p53, a series of serine/threonine protein                (Smad 2, 3, and 4), thereby inhibiting their ability to bind to DNA
kinases (i.e., the cyclin-dependent kinases or CDKs) that control                 (25). Likewise, E7 is known to specifically interact with the Rb tumor
the passage of the cell through the various phases of the cell cycle,             suppressor protein. Within the G1 phase of the cell cycle, Rb forms
the regulatory subunits (cyclins) that control CDK activity, and the              a complex with the E2F transcription factor and prevents E2F from
corresponding CDK inhibitors (p16INK4A, p21-Waf-1, and p27KIP). A                 activating gene transcription. At the G1/S boundary, the Rb protein
detailed review of the cell cycle is beyond the scope of this article,            is phosphorylated with release of the E2F transcription factor—
and the reader is directed to the appropriate references for a                    thereby initiating E2F gene transcription and entry into the S phase
detailed description (20,21).                                                     of the cell cycle. The HPV E7 oncoprotein abrogates this control
    The E6 and E7 oncoproteins of the high-risk HPV subtypes                      mechanism by directly binding with Rb and displacing E2F from the
have been implicated in a number of cellular processes related to                 complex. This results in E2F-driven gene transcription independent
increased proliferation and neoplastic transformation of the infected             of normal cell cycle control (22). This release of E2F uncouples
keratinocytes. The E6 protein has been implicated in two critical                 gene transcription from cell cycle control and results in prolonged
processes. The first is the degradation of the p53 tumor suppressor               and aberrant transcription of S-phase genes responsible for DNA
protein through ubiquitin-mediated proteolysis. Removal of                        synthesis and cellular proliferation. In addition, the combined actions
functional p53 eliminates a major cell cycle checkpoint responsible               of both E6 and E7 have been shown to contribute to centrosomal
for DNA repair prior to entry into DNA replication and mitosis (22).              abnormalities and subsequent genomic instability in CN (26). These
In addition, E6 has been shown to interact with the c-myc protein                 effects are summarized in Figure 3.
and is responsible for direct transcriptional activation of the hTERT
gene with subsequent expression of telomerase (23,24). Activation                 CYCLINS AND CN
of telomerase is a key step in cancer biology, responsible for the
                                                                                      In CN, numerous alterations in the cell cycle have been identified
maintenance of telomere length on replicating chromosomes, and
                                                                                  as a result of HPV infection. These alterations include abnormal
this enzyme ensures functionally intact chromosomes during cellular
immortalization. The HPV oncoprotein E7 is known to contribute to                 overexpression of various cyclins (including cyclins A, B1, and E)
cellular proliferation through two independent mechanisms. The                    and overexpression of various CDK inhibitors (e.g., p16INK4A). The
first is the inactivation of the transforming growth factor-β (TGF-β)             general alterations in the cell cycle that accompany CN are shown
tumor suppressor pathway responsible for cell cycle arrest at the                 in Figure 3. These alterations have been investigated for potential


                                                                                                                       TGF beta tumor
                                                                                                                     suppressor pathway
                                                                        G1
                                          HPV E7

                                                                     Rb / E2F                                            Smad 2,3,4
                                                      Cyclin D +               Cyclin E                                                        HPV E7
                                                      CDK 4,6                  increased
                                                                                    E2F release
                                                          p-Rb                      from Rb
                                                                                                                         Abrogates G1 arrest
                                                                                           Abrogates G1/S
                                                                                             check point
                                                                                                                         Aberrant S-phase Induction
                                                Cyclin A, B
                           M                    + CDK1                                          Cyclin A +         E2F transcription of S-
                                                                                                CDK2               phase genes
                                                                        Cyclin B +                                 DNA replication
                                                                        CDK1                                S
                      Abrogates G2/M                                                                              Telomerase expression
                        check point


         HPV E6                          p53
                                                                                                                   hTERT transcription

                           Mdm 2                                       G2
                                                                                              HPV E6                                        �����


Figure 3. Cell cycle alterations and proliferation control defects in cervical neoplasia (CN). Human papillomavirus (HPV) infection and overexpression of the
E6 and E7 oncoproteins produces a series of alterations in the cell cycle and proliferation control. The HPV E6 oncoprotein abrogates cell cycle checkpoints at
the G1/S and G2/M boundaries with subsequent replication of DNA with somatic mutations. E7 promotes the acceleration into the S-phase with prolonged ex-
pression of their S-phase genes required for DNA replication (aberrant S-phase induction). Likewise, E6 promotes expression of telomerase ensuring continued
chromosomal telomere integrity during proliferation and cellular immortalization. Finally, E7 abrogates the transforming growth factor-β (TGF-β) signaling
pathway and abrogates this control mechanism for G1 arrest and control of proliferation. CDK, cyclin-dependent kinases.

April 2005                                                                                                                Biomarkers in Cancer Research     19
Table 2. Cyclins in Cervical Neoplasia
                         Cell Cycle
 Cyclin Protein           Phase                        Function                                   Expression Status in CN                       References
 Cyclin D1 and D3      G1             CDK4 and CDK6 regulatory subunits.             Cyclin D1 decreased in 97% of HSIL and 72% of              27,30–32
                                      Links mitogenic stimuli to cell cycle          invasive cervical cancer.
                                      progression.                                   Cyclin D3 decreased in 51% of squamous cell
                                                                                     carcinoma.
                                                                                     Increased expression within cervical carcinoma
                                                                                     associated with poor disease-free survival.
 Cyclin E              G1             CDK2 regulatory subunit.                       Increased in 97% of LSIL, 92% of HSIL, and 82%             27–29
                                                                                     of squamous cell carcinoma.
 Cyclin A              S and M        CDK2 regulatory subunit in the S-              Increased in 35% of squamous cell carcinoma                27,33
                                      phase and CDK1 in the M-phase.                 and adenocarcinoma.
                                      Links anchorage-dependent cell
                                      growth to cell cycle progression.
 Cyclin B              G2 and M       CDK1 regulatory subunit.                       Increased in CN, squamous cell carcinoma, and              27,33
                                                                                     adenocarcinoma.
 CN, cervical neoplasia; CDK, cyclin-dependent kinases; HSIL, high-grade squamous intraepithelial lesions; LSIL, low-grade squamous intraepithelial lesion.




Table 3. Characteristics of p16INK4A and MCM-5 Staining of Cervical Neoplasia
 Features                             Characteristics of p16 Staining                            Characteristics of MCM-5 Staining

 Sensitivity for HSIL detection       70%–100%                                                   100%
 Specificity for HSIL detection       Low and inconsistent: 25%–75%                              67%
 Scoring criteria                     Variable across studies with a minimum of 10+              Percent positive cells detected in tissue biopsy
                                      positive cells per slide                                   specimens.
 Staining localization                Overexpression in both the nucleus and the                 Staining localized to nucleus.
                                      cytoplasm.
 Specificity of staining              Expression in both nucleus and cytoplasm makes             Nuclear localization makes immunocytochemistry
                                      immunocytochemistry interpretation difficult.              interpretation easy.
                                      Expression detected in both LSIL and HSIL
                                      lesions.                                                   Expression detected in proliferative cells within
                                                                                                 parabasal layer within cervical epithelium.
                                                                                                 Easy detection of neoplastic cells within tissue
                                                                                                 section lesions.
                                                                                                 Difficult discrimination between basal cells and
                                                                                                 malignant cells within cervical cytology preparations.
 MCM-5, minichromosome maintenance 5; HSIL, high-grade squamous intraepithelial lesions; LSIL, low-grade squamous intraepithelial lesion.




utility in the clinical diagnosis of high-grade cervical disease. Cyclin           has been examined and is associated with carcinoma, biopsy
E, A, and B have all been shown to be overexpressed in squamous                    confirmed CIN 2+ lesions, and a significant number of LSIL—CIN 1
cell cervical neoplasia as well as in LSIL and HSIL lesions (26–32).               lesions (41–44). The p16 protein can be detected in both histology
Likewise, cyclins A and B have been shown to be overexpressed                      specimens as well as liquid-based cytology (39–41). Characteristics
in cervical adenocarcinoma and its malignant precursors (33). To                   of p16 detection of cervical disease are summarized in Table 3, and
date, none of the cyclins that are overexpressed in HPV-induced                    the clinical performance of p16 detection of HSIL specimens within
neoplasia display sufficient sensitivity or specificity for the accurate           cytology specimens is shown in Table 4.
detection of high-grade cervical disease (either for CIN2+ lesions
in histology or HSIL+ cells in cytology) to be useful as independent               MINICHROMOSOME MAINTENANCE PROTEINS
molecular markers in a clinical diagnostic assay. Moreover, over-                  AS MARKERS OF CELLULAR PROLIFERATION
expression of cyclin E appears to be a surrogate marker for HPV
                                                                                       The minichromosome maintenance (MCM) proteins function
(28). These published observations are summarized in Table 2.
                                                                                   in the early stages of DNA replication through loading of the pre-
                                                                                   replication complex onto DNA and functioning as a helicase to help
p16INK4A AND CN                                                                    unwind the duplex DNA during de novo synthesis of the duplicate
    p16INK4A (p16) is an inhibitor of CDKs 4 and 6 and functions in                DNA strand. Early publications have shown that the MCM proteins,
the progression from G1 to S phase of the cell cycle. In response                  and in particular, MCM-5, are useful for the detection of cervical
to infection by high-risk HPV infections, p16 is overexpressed in                  disease (45) as well as other cancers (46). The published literature
CN including HSIL lesions and carcinoma (34). Overexpression of                    indicates that antibodies to MCM-5 are capable of detecting CN
p16 has been shown to correlate with HPV type 16 and 18 infec-                     cells. The specificity for detection of high-grade cervical disease has
tions and can be detected in both squamous cell carcinoma and                      not been demonstrated for MCM-5 (45). The detection of MCM-5
adenocarcinoma (35–40). The specificity of p16 overexpression                      expression is not restricted to high-grade cervical disease but is

20   Biomarkers in Cancer Research                                                                                                                    April 2005
Table 4. Performance of p16INK4A and MCM-5 Detection of                             HPV viral subtypes and the molecular behavior of cervical disease.
HSIL in Cervical Cytology Applications                                              Furthermore, overexpression of the MCM proteins is not restricted
  Marker            Sensitivitya         Specificityb            Reference          to cervical carcinoma, but is known to be a characteristic of other
                       (%)                  (%)                                     neoplasias (46). As such, this aberrant expression of genes
                                                                                    controlled by E2F and the resulting increase in S-phase induction
  p16INK4A               96                    33                     39
                                                                                    appears to be a common characteristic of many cancers.
  p16INK4A               92                    53                     40
  p16INK4A               97                    42                     41
                                                                                    EMERGING DIAGNOSTIC ASSAYS FOR THE
  MCM-5                  100                   67                     43
                                                                                    DETECTION OF CERVICAL DISEASE
  Sensitivities and specificities for the detection for HSIL in cervical cytology
  calculated from the data in the referenced literature. MCM-5, minichromo-             The application of transcriptional profiling using DNA micro-
  some maintenance 5; HSIL, high-grade squamous intraepithelial lesions;            arrays has identified a number of genes that are overexpressed
  LSIL, low-grade squamous intraepithelial lesion; TP, true positive (HSIL          within cervical disease samples. Genes overexpressed in cervical
  stained with biomarker); FP, false positive (LSIL stained with biomarker);
  TN, true negative (LSIL not stained with biomarker); and FN, false negative       carcinoma and in response to HPV infections have been described
  (HSIL not stained with biomarker).                                                in the literature (50–52). Using this approach, MCM-6, MCM-4,
   Sensitivity = TP/TP + FN.                                                        and TOP2A have been identified as overexpressed genes in CN
  a
  b
   Specificity = TN/TN + FP.
                                                                                    (50–52). In addition, other promising candidates have been identified
                                                                                    by DNA microarray profiling, including IGFBP-3 and claudins 1
also detected in identified low-grade dysplasia and proliferative                   and 7. Translational research on the genes identified using these
cells that have re-entered the cell cycle following infection with                  approaches has yielded interesting advances in both the under-
high-risk HPV. The detection of CN with antibodies to MCM-5 is                      standing and the application of these disease-specific markers for
shown in Figure 4. In addition to MCM-5, other members from the                     the detection of cervical disease (48,49,53–55). Preliminary results
MCM family, including MCM-2 and MCM-7 have been shown to be                         have shown that the genes identified from the microarray analysis,
potentially useful markers for the detection of CN in tissue samples                such as MCM-6 and TOP2A, encode proteins that are over-
(46,47). Table 4 summarizes the published performance of MCM-5                      expressed in cervical disease (48,49). These proteins have been
in the detection of cervical disease. Recent results have shown that                used to develop molecular immunohistochemistry and immunocy-
MCM-7 appears to be a specific marker for the detection of high-                    tochemistry assays for the detection of high-grade cervical disease.
grade cervical disease using immunochemistry formats (47–49).                       These markers have been reported to detect cervical disease with a
                                                                                    higher level of specificity and positive predictive value over current
EMERGING MARKERS AND THE MOLECULAR                                                  methods of HPV detection or cytology-based diagnosis (48,49).
DESCRIPTION OF CN                                                                   The detection of cervical cancer in a cytology specimen using one
    Molecular markers such as MCM-2, MCM-7, and MCM-6 hold                          such prototype molecular immunocytochemistry assay is shown in
promise for the development of more specific assays to detect                       Figure 5.
cervical disease. In addition to these markers, topoisomerase II-α                      It is anticipated that further development and investigation of
(TOP2A) has been shown to be overexpressed in cervical disease                      molecular assays employing markers such as the MCM proteins
and to correlate with the detection of aberrant S-phase induction                   and TOP2A within clinical settings will define the clinical utility of
and transcriptional activation present within CN (48,50). As shown                  these molecular-based diagnostics for the specific improvement in
in Figure 3, the action of the HPV oncoproteins E6 and E7 abrogate                  the detection of cervical disease. As stated previously in this article,
the critical cell cycle checkpoints and induce expression of S-phase                the dilemmas of morphology-based diagnosis will benefit from
genes through the constitutive action of the E2F transcription factor.              the development and use of more objective molecular diagnostic
This results in prolonged and active induction of S-phase genes                     assays based upon discrete molecular changes in response to HPV
and DNA replication outside the normal control mechanisms of                        infection and subsequent neoplastic transformation. The use of
the cell cycle. Key proteins such as the MCM proteins and TOP2A
are expressed during this aberrant gene transcriptional activation.
This behavior appears to be a hallmark within high-grade cervical
disease and provides the link between infections with oncogenic




             CIN 3 100×                                     HSIL 400×

Figure 4. The detection of minichromosome maintenance 5
(MCM-5) overexpression in cervical high-grade intraepithelial
neoplasia using specific monoclonal antibodies in an immuno-
chemistry format. MCM-5 overexpression is detected in both tissue
(left panel) and cytology specimens (right panel) using a molecular                 Figure 5. The detection of an invasive cervical cancer cell using a molecular
immunochemistry format (48,49). The overexpressed MCM-5 protein is                  immunocytochemistry assay in a routine SurePath liquid-based cytology
localized to the nucleus of the neoplastic cells. CIN3, cervical intraepithelial    specimen. Overexpressed proteins are detected using monoclonal antibodies
neoplasia grade 3.                                                                  in an immunocytochemistry format (48,49).



April 2005                                                                                                                  Biomarkers in Cancer Research     21
these molecular descriptors of CN is anticipated to permit the devel-                        J.L. Walker, G.A. Johnson, et al. (ASCUS-LSIL Triage Study Group). 2003.
opment of objective molecular assays to detect high-grade cervical                           Results of randomized trial on the management of cytology interpretations of
                                                                                             atypical squamous cells of undetermined significance. Am. J. Obstet. Gynecol.
disease. The development of such objective clinical diagnostics that                         188:1383-1392.
specifically detect cervical disease without relying on the subjective                   10. Schiffman, M., D. Solomon, R. Tarone, E.E. Partridge, L. Kilgore, S. Hester,
interpretation of an individual cytopathologist will represent                               J.L. Walker, G.A. Johnson, et al. (ASCUS-LSIL Triage Study Group). 2003. A
a significant advancement in the clinical detection of cervical                              randomized trial on the management of low-grade squamous intraepithelial lesion
disease. Furthermore, the ability to use currently available and                             cytology interpretations. Am. J. Obstet. Gynecol. 188:1393-1400.
                                                                                         11. Guido, R., M. Schiffman, D. Solomon, and L. Burke. 2003. Postcolposcopy
standardized liquid-based cytology specimens in conjunction with                             management strategies for women referred with low-grade squamous intraepi-
emerging molecular diagnostics promises to improve the screening                             thelial lesions or human papillomavirus DNA-positive atypical squamous cells of
and detection of cervical disease and to significantly improve the                           undetermined significance: a two-year prospective study. Am. J. Obstet. Gynecol.
current state of cervical disease screening and diagnosis within the                         188:1401-1405.
healthcare system.                                                                       12. Cox, J.T., M. Schiffman, and D. Solomon. 2003. Prospective follow-up suggests
                                                                                             similar risk of subsequent cervical intraepithelial neoplasia grade 2 or 3 among
                                                                                             women with cervical intraepithelial neoplasia grade 1 or negative colposcopy and
HPV VACCINE DEVELOPMENT                                                                      directed biopsy. Am. J. Obstet. Gynecol. 188:1406-1412.
    Recent work in the development of vaccines for the prevention                        13. Manos, M.M., W.K. Kinney, L.B. Hurley, M.E. Sherman, J. Shieh-Ngai, R.J.
                                                                                             Kurman, J.E. Ransley, B.J. Fetterman, et al. 1999. Identifying women with cer-
of HPV infection in young patients has been demonstrated for                                 vical neoplasia. Using human papillomavirus DNA testing for equivocal Papanico-
HPV types 16 and 18 (56–58). The efficacy of such vaccinations                               laou results. JAMA 281:1605-1610.
is anticipated to significantly improve public health with the ultimate                  14. Wright, T.C., M. Schiffman, D. Solomon, J.T. Cox, F. Garcia, S. Goldie,
goal of eliminating cervical cancer on a global basis. The implemen-                         K. Hatch, K.L. Noller, et al. 2004. Interim guidance for the use of human
tation of such a vaccination program will require measurement of                             papillomavirus DNA testing as an adjunct to cervical cytology for screening.
                                                                                             Obstet. Gynecol. 103:304-309.
both effective clinical end points for vaccine efficacy as well as an                    15. Vassilakos, P., F. de Marval, M. Munoz, G. Broquet, and A. Campana. 1998.
active monitoring program post-vaccine introduction to insure that                           Human papillomavirus (HPV) DNA assay as an adjunct to liquid-based Pap test
the vaccination program is working. The use of highly sensitive                              in the diagnostic triage of women with an abnormal Pap smear. Int. J. Gynecol.
analytical methods for the detection of HPV, such as PCR-based                               Obstet. 61:45-50.
detection and genotyping methods, as well as emerging molecular                          16. Lisell, C.L., S. Setty, H.E. Gulbahce, D.M. McKeon, C.A. Brown, R.C. Mc-
                                                                                             Glennen, J. Rohlader, and S.E. Pambuccian. 2003. Polymerase chain reaction
diagnostics for the detection of active CN would be useful clinical                          (PCR)-based reflex HPV testing is an effective ASCUS triage method in teenag-
tools in the monitoring of HPV vaccination programs on a global                              ers. Acta Cytol. 47:7.
basis. The markers discussed in this article and the emerging                            17. Avissar, P.L., H. Li, and D.P. Malinowski. 2003. Genotyping analysis of HPV in
molecular diagnostic assays utilizing these markers are expected                             cervical carcinoma within Vietnamese women. Acta Cytol. 47:28.
to become important clinical tools in patient monitoring and surveil-                    18. Bolick, D. 2003. HPV genotyping from SurePath™ pap specimens by DNA
                                                                                             sequencing. Acta Cytol. 47:29.
lance programs related to a global HPV immunization effort.                              19. Avissar, P.L., M.D. Harrison, H. Li, and D.P. Malinowski. 2004. Adequacy of
                                                                                             SurePathTM cytology samples for DNA-based applications: HPV detection, identi-
ACKNOWLEDGMENTS                                                                              fication and quantitation. Acta Cytol. 48:698.
   The author would like to thank Timothy J. Fischer, Adriann J.                         20. Kastan, M.B. and J. Bartek. 2004. Cell-cycle checkpoints and cancer. Nature
Taylor, and Margaret R. Parker for helpful discussions and for                               432:316-323.
providing the microphotographs shown in Figures 4 and 5.                                 21. Massague, J. 2004. G1 cell-cycle control and cancer. Nature 432:298-306.
                                                                                         22. Duensing, S. and K. Munger. 2003. Centrosome abnormalities and genomic
COMPETING INTERESTS STATEMENT                                                                instability induced by human papillomavirus oncoproteins. Prog. Cell Cycle Res.
                                                                                             5:383-391.
    The author is employed by Tripath Imaging, Inc., which manufac-                      23. McMurray, H.R. and D.J. McCance. 2003. Human papillomavirus type 16 E6
tures and sells medical devices, instruments, and diagnostic assays                          activates TERT gene transcription through induction of c-myc and release of
related to cancer screening, detection, and diagnosis. Dr. Malinows-                         USF-mediated repression. J. Virol. 77:9852-9861.
ki has stock/stock options in Tripath Imaging, Inc.                                      24. Veldman, T., X. Liu, H. Yuan, and R. Schlegel. 2003. Human papillomavirus
                                                                                             E6 and myc proteins associate in vivo and bind to and cooperatively activate the
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