The Molecular Pap: E6 and E7 Hold Promise for Cervical Cancer Screening

Molecular testing offers great promise in detecting cellular abnormalities in women whose DNA has been transformed and, therefore, in identifying women at greatest risk of having or developing cervical cancer.  How might a new molecular test for E6, E7 affect current cervical cancer screening strategies?

Currently, our basic strategies for cervical cancer screening can be summarized as follows:

1. Exclude CIN 2-3 (HSIL)
2. Determine the risk
3. Rule out invasion
4. Establish the next screening interval

The first two strategies listed here are often interchangeable in their occurrence, and our means of accomplishing these steps are summarized in Table 1.

Screening Step	Action/Means/ Considerations	Notes
Determine the risk	Age Pap Test (HPV test)	Women under 21 are not tested for HPV by ASCCP guidelines Clinical risk factors (multiple sexual partners, smoking, etc.) are not incorporated into ASCCP guidelines Positive HPV test neither confirms nor excludes possibility of HSIL (CIN 2-3) hrHPV testing evaluates for HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 Typing for HPV subtypes 16, 18 is a separate means of risk stratification testing HPV testing for LSIL not recommended by ASCCP
Exclude CIN 2-3	Routine Pap testing (or colposcopy) Age	Pap tests results are very specific, but lack sensitivity HPV+ women over 30 y/o fall into higher risk category
Rule out invasion	Colposcopy	Biopsies of abnormal areas to exclude CIN 2-3 and invasion
Establish the next screening interval	Inform patient of when next cervical collection will occur	ASCCP guidelines used for determining next interval

Table 1

Hypothetical 25 y/o Patient

Let’s use a hypothetical 25 year old patient with ASC-US Pap results to illustrate the process outlined in Table 1.

1) Exclude CIN 2-3+

Our hypothetical patient has an abnormal Pap test (ASC-US) for which CIN 2-3 is not excluded.

The ASC-US – LSIL Long Term Follow-Up Study (ALTS) found that women having ASC-US Pap results AND who have a positive high risk HPV (hr HPV) test were found to have a significant CIN 2-3 cervical lesion on colposcopic follow-up in approximately 10-20% of patients.  Therefore, the American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines specify colposcopic evaluation to rule out the presence of high grade squamous intraepithelial lesion or invasive carcinoma in patients having ASC-US Pap results AND a positive HPV test.

2) Determine the Risk

If our patient, age 25, had a normal Pap test, she would return in 1 or 2 years for her next routine Pap depending on which interval an office chooses to follow.

For our hypothetical patient, however, the Pap test has Atypical Squamous Cells of Undetermined Significance (ASC-US), and a reflex HPV test is now performed to assess the patient’s risk category.

An abbreviated algorithm followed for this patient looks like this:

It is important to understand that the HPV test is a risk stratification test, not a test for cervical cancer or its precursors.  Positive HPV results signify nothing more than placement of the patient into a higher risk category.  It is the patient’s risk category that helps stratify the approach to the next steps in her care.

Negative HPV results would place the patient into a category having the same risk as patients with a normal Pap test.  Hence, a patient with negative hrHPV testing would return to the “normal pool,” almost as if she had never had an abnormal Pap result to begin with.

3) Rule Out Invasion

The Pap test can report squamous carcinoma when specific cytologic criteria are present, but the gold standard to exclude invasive disease is colposcopy with directed biopsy of abnormal-appearing regions of the cervix.

4) Establish the Next Interval

For our patient, if her hrHPV test was negative, she would return at the routine interval for her next Pap test.  If our patient had positive reflex hrHPV testing, colposcopy is recommended to rule out 2-3+.  Cervical biopsies positive for high grade dysplasia or invasive carcinoma would initiate therapeutic intervention.

For our hypothetical patient, her colposcopy and biopsy are negative and she is instructed to return in one year for repeat Pap test and hrHPV testing per ASCCP algorithmic guidelines.

Discussion

It is important to note that the HPV test is a risk stratification test that, by helping establish risk category, adds sensitivity to the routine Pap testing protocol and helps determine which patients should undergo colposcopy.  There are evidence-based algorithms for ASC-US and the results of subsequent reflex hrHPV testing.

Not all patients with HPV infection will develop invasive cervical cancer.

Important question: If not every woman with HPV infection will develop invasive cancer, why do we currently approach LSIL and ASC-US abnormalities as if the patient could have invasive cancer?

Answer:  Given evidence-based studies and existing technology, this approach represents our current state-of-the-art strategy.  Colposcopy is performed following LSIL/ASCUS, +HPV Pap results to rule out CIN 2-3+ and invasive disease.

The Wish For A Better Test

Future testing will need to focus on identifying women who are most likely to have potentially lethal cervical disease.  Stated differently, we need a test that will separate those women having mere transient HPV infection and its associated non-lethal Pap and biopsy abnormalities from those having HPV-driven neoplasia.

The Better Test May Be Here

Integration by HPV into the host’s DNA is a key step in neoplastic transformation (see Glossary). An exciting new molecular approach in identifying transformed cells uses the principle of identifying two HPV gene products, E6 and E7, that are overexpressed in cells where HPV has integrated into the host genome and has initiated neoplasia (see Glossary).

Review of the Role of HPV Infection in Cervical Cancer

Cellular Infection

HPV invades the skin or mucosa by entering tiny breaks in the surface (even those not visible to the naked eye). Once inside, HPV infects host epithelial cells, tricking them into producing new viruses. Then, in the process of normal cell replacement, the infected cells die, releasing viral particles.

High risk strains of HPV can integrate their viral DNA into the host’s DNA.  This is not a normal part of the HPV life cycle and is felt to occur in only a minority of HPV infections.  Viral integration, however, may give infected host cells a selective advantage, leading to a longer infection time and, in the case of malignant transformation, cellular immortality.  The longer the infection lasts, the more time there is for integration and abnormal cellular events to develop.

The HPV genome contains several genes that encode proteins.  In the HPV genome, three of these genes — E2, E6, and E7 — are of particular interest because of their roles in the development of cervical cancer. The E2 protein functions by binding to both the E6 and E7 proteins. When E6 and E7 are bound to E2, they are blocked from their normal actions in the cell.

Oncoproteins E6 & E7 Block Production of Two Suppressor Proteins: p53 & Rb

Overview

After HPV integration into the host’s DNA, the viral genes E6 and E7 may be over-expressed, creating proteins that prevent the activity of key tumor suppressors.  Here, E6 can inhibit p53, a protein that controls responses to different types of cellular stress, including DNA damage and viral infection (see Glossary).  The E7 protein can inhibit Rb, a protein that can prevent cell division by blocking the activity of transcription factors.

The combined effects of E6 and E7, therefore, can put cells at risk for undergoing uncontrolled division that can lead to cancer.

E6 – Can Bind p53

When E6 is not bound to E2, it is free to bind to the p53 tumor suppressor protein. Tumor suppressors are normal occurring anti-cancer proteins.  When E6 binds to p53, p53 is destroyed and cannot function.  The p53 protein is a key element in cell cycle control and is not functional in over 50% of all human cancers. Without the p53 protein, a cell has reduced ability to respond to DNA damage and may continue to divide even if it is damaged (see Glossary).

The E6 protein causes the expression of telomerase, a protein that is not normally produced by most  cells in adult humans. When present, telomerase maintains the ends of the chromosomes. This prevents the breakdown of chromosomes and helps cancer cells to divide unchecked — essentially forever.

Since the expression of E6 is strictly required for maintenance of a malignant phenotype in HPV-induced cancers, the E6 proteins are a target of therapeutic HPV vaccines.

E7 – Can Bind Rb

The HPV protein E7 also plays a key role in helping HPV manipulate control of infected cells by its antagonism of a cellular transcription factor, E2F, which causes the cell to proceed along the cell cycle and to divide.  When not bound by E2, E7 binds to the retinoblastoma protein (Rb). When E7 is bound to Rb, Rb cannot carry out its normal function, which is to bind to E2F, the transcription factor that causes cell cycle progression.

When bound to Rb, E2F cannot function as a transcription factor and, therefore, cannot help the cell to divide. But, if E7 binds to Rb, E2F then cannot bind to Rb, and is thereby free to act unchecked as a transcription factor, a critical step in cancer development.

In essence, E7, by binding to Rb, “inhibits an inhibitor” of cell division, thereby preventing cell death (apoptosis) and, subsequently, promoting cell division (see Glossary).  E7 also participates in immortalization of infected cells by activating cellular telomerase.

Simply stated, the E6 and E7 proteins help HPV hijack cell division to become immortal and to help drive cellular neoplasia.

E6 E7 mRNA – A Molecular Probe Test

One example of a new molecular test developed to detect transformed cells is HPV OncoTect^TM E6, E7 mRNA Kit, a product of the InCell Dx company.  Although not an endorsement by InCyte Pathology, the following promotional literature from InCell Dx highlights the exciting capabilities of molecular testing which is available to be performed on Pap collections.

The HPV OncoTect^TM E6, E7 mRNA Kit is a unique detection method that measures both the number of transforming cells and the quantity of E6, E7 mRNA in each cell.  These two measurements precisely assess the overexpression of E6, E7 mRNA in routine patient samples collected in ThinPrep® and Surepath^TM vials, thereby further refining accuracy and specificity of HPV testing.

Many women with positive test results from a HR HPV DNA Test will have normal biopsy. The HPV OncoTectTM E6, E7 mRNA Test is only positive if there is overexpression of E6, E7 — a sign of neoplastic transformation. In the life cycle of the human papillomavirus, the overexpression of E6, E7 mRNA in a cell is the molecular switch leading to cervical cancer.

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SUMMARY

Molecular testing offers great promise in detecting cellular abnormalities in women whose DNA has been transformed and, therefore, in identifying women at greatest risk of having or developing cervical cancer.  This new testing employs detection of gene products E6 & E7, a molecular approach that provides greater specificity to current cervical cancer screening strategies.

Our Hypothetical Patient

How might a test for E6, E7 affect our hypothetical patient with the ASC-US Pap test?  If molecular testing for transformed cells lives up to its promise in the future, an ASC-US result for our patient may dictate E6, E7 testing performed instead of hrHPV testing, and there are those who are discussing that this type of testing (or similar molecular approaches) could replace the Pap test altogether.  Seems premature, but there is no question that molecular testing will have impact on the ever-evolving strategies in cervical cancer testing.  In the meantime, we employ ASCCP guidelines and the four basic strategies that we have discussed.  Stay tuned.

 GLOSSARY OF TERMS

Term	Definition
Early HPV proteins	The HPV genome is composed of six early (E1, E2, E3, E4, E6, and E7) and two late (L1 and L2) genes.  Majority components of the exterior shell that surrounds the circular HPV DNA are coded for by the ‘E’ genes of the HPV genome.
Late HPV proteins	The HPV genome is composed of six early (E1, E2, E3, E4, E6, and E7) and two late (L1 and L2) proteins are keystone corners of the decahedron shell that surrounds the circular HPV DNA and are coded for by the ‘L’ genes of the HPV genome.
upstream regulatory region (URR)	HPV genomes isolated from cervical cancers often contain nucleotide sequence alterations in the HPV upstream regulatory region (URR) that controls viral-gene transcription and drives viral replication.
neoplasia	Neoplasia is abnormal and irreversible proliferation of cells. The growth of neoplastic cells exceeds and is not coordinated with that of surrounding normal tissues. Abnormal growth persists in the same excessive manner even after cessation of stimuli. Neoplasia often causes a lump or tumor. Neoplasms may be benign, pre-malignant (carcinoma in-situ), or malignant (cancer).
telomerase	Telomerase is an enzyme that adds DNA sequence “repeats” to the end of DNA strands in the telomere regions, which are found at the ends of eukaryotic chromosomes. Telomerase consists of repeated nucleotides containing non-coding DNA material.  Telomerase prevent constant loss of important DNA from chromosome ends.
transformation	Malignant transformation is the process by which cells acquire the properties of cancer. This may occur as a primary process in normal tissue, or secondarily as malignant degeneration of a previously existing benign tumor.
integration	In viral infection, integration refers to incorporation of viral genetic material into the host’s DNA.
tumor promoters	Tumor promotion is a process in which existing tumors are stimulated to grow. It is the second phase in tumor development. Tumor promoters by themselves are not able to cause tumors to form.  In molecular biology, tumor promoter regions are genes that code for proteins that promote cellular or neoplastic growth.
tumor suppressors	A tumor suppressor gene, or anti-oncogene, is a gene that protects a cell from steps on the path to malignant transformation. When a tumor suppressor gene is mutated to cause a loss or reduction in its function, the cell can progress to neoplasia, usually in combination with other genetic changes.

                               

Term	p53 & Rb Proteins  -  Definitions and Notes
p53 protein	p53 is a tumor suppressor protein.  It is crucial in multicellular organisms, where it regulates the cell cycle and functions as a tumor suppressor. As such, p53 has been described as “the guardian of the genome”, the “guardian angel gene”, and the “master watchman”, referring to its role in conserving stability by preventing genome mutation.  The name p53 is a reference to its molecular mass.  When bound to E6, p53 is destroyed and cannot function.
Rb protein	Retinoblastoma (Rb) protein is a tumor suppressor protein that is dysfunctional in many types of cancer.  One highly studied function of Rb is to prevent excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide.  It is also a recruiter of several chromatin remodeling enzymes such as methylases and acetylases.  In humans, the protein is encoded by the Rb1 gene.  Early studies of Rb1 showed that, if both alleles of this gene are mutated early in life, the protein is inactivated, thereby contributing to the development of retinoblastoma cancer, and hence, the name Rb.  It is not known why an eye cancer results from a mutation in a gene that is important everywhere in the body.   Rb prevents cells from replicating damaged DNA by preventing progression along the cell cycle through G1 (first gap phase) into S (synthesis) phase.  Rb binds and inhibits transcription factors of the E2F family, which are composed of dimers of an E2F protein and a dimerization protein termed “DP”.  The transcription activating complexes of E2 promoter-binding-protein-dimerization partners (E2F-DP) can push a cell into S phase.  As long as E2F-DP is inactivated, the cell remains stalled in the G1 phase.  When Rb is bound to E2F, the complex acts as a growth suppressor and prevents progression through the cell cycle.  The RB-E2F/DP complex also attracts a histone deacetylase (HDAC) protein to the chromatin, reducing transcription of S phase promoting factors, further suppressing DNA synthesis.  If E7 (instead of E2F) is bound to Rb, E2F acts unchecked as a promoter of cell growth.

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