E1: act on origin of replication to induce episomal DNA replication.
E2: together with E1 forms origin binding complex. Also a transcription regulator 
Particularly a repressor of the E6 promoter.
E4: Act with intermediate filaments of host cell, associate wih viral particle release.
E5a: Anoncoprotein related to the signal transduction of host cell. Act on the ER,
Golgi, related to the endocellular trafficking.
E6: Anoncogene, inactivation of p53 tumor suppresser gene.
E7: An oncogene, inactivation of Rb tumor suppresser gene.
E7 gene alone of either low or high risk HPV transactivate host DNA replication 
In the differention of squamouse epithelium.
Differentiation of host cell is required to turn on the E6/E7 promoter and hence
Induction of host DNA replication.
L1: External capsid protein of the hexahedral capsid.
L2: Internal capsid protein of the hexahedral capsid.

1.Epidemiology:
     There are 5000,000 new cases of cervical or anal cancer each year around the world, which are related with the HPV infection. Cervical cancer has been the leading prevalent cancer in Taiwan since 1985. HPV 16, 18 are related to the development of squamous, adeno-and small cell carcinoma of cervij, avd metastasis of the cervical cancer.

PHYLOGENETIC TREE OF HPV BASED ON AMINO ACID SEQUENCES

2.Patholphysiology:
(A) HPV infection
      The primary infection site of HPV is at the squamous-columnar(S-C) junction of 
Cervical epithelium, where the cells are actively proliferating. The columnar epithelial cells at the S-C junction toeard the endocervix with age. Most cervical cancer occurs at the region between the new and old S-C junctions, which is therefor named “transforming zone”. The original S-C junction of a teenage girl is more exposed at the exocervix and cells within are more vulnerable to HPV infection if she has early sexual exposure. Other estrogen-excess status that leads to cervical extropian and vulnerability include pregnancy and use of oral pills. The way of HPV entry into the epithelium is unknown, probably is through injured (erotic) or inflamatory epithelium and get into the basal (stem) cells.

HPV  Transmission

•  Cutaneous HPVs are transmitted casually.

•  Genital HPVs are transmitted sexually.

         Important variables for acquisition:

          － Age at first intercourse

          － Number of sex partners

          － Coincident STDs

          － Immunc status

(B)Integration of HPV DNA into host genome

     HPV DNA is usually extrachromosomal in CIN lesions. In malignment lesion Viral DNA is usually found to be integrated. The integration disrupt HPV circular genome at the E2 gene which is a repressor for E6/E7 promoter. This results in an over-expression of E6 and E7 oncogenes. No specificity has been observed for intrachromosomal localizations of HPV integration. One report disclose HPV 16 integrated near the c-src, c-raf and c-myc oncogenes. A small percentage of malignant tumors harbors only nonintegrated copies of viral DNA.

     The integration is clonal in nature indicating the HPV infection precedes the tumor growth. The infection of HPV is tissue (i. e. keratinocyte) specific. The abundant AP-1 in Keratinocyte but not fibroblast may be responsible, since there is a crucial AP-1 binding site at the E6 promoter.

B) E6 and E7 oncogenes
    The vast majority of HPV-positive cancer cells express high level of E6 and E7.
E6, E7 genes from high risk HPV together are capable of immortalizing primary human keratinocytes, subsequent transfection with v-ras, or after long-time culture, may lead to malignant clones.
     Protein products of deregulated E6 and E7 genes bind to tumor repressor gene products,p53 and pRb, and leads to loss of function of these tumor suppressor proteins.p53 and pRb, are also bound to other viral oncoproteins such as E1b (p53) and E1a (pRb) of adenovirus, and large T antigen (both) of SV40 virus.

Expression of the high risk HPVs may lead to aneuploidy (change of chromosome number) and contribute to the other cellular events necessary for a full cancer development.
E7 protein only is sufficient to turn on the DNA replication machinery. E7 binds 
Rb protein, ieading to its sequestration to an inactive form. The pRb normally activate the transfection factor E2F, which is a major machinery to activate the expression of a lot of replication related enzymes and entry into the cell cycle.
The major role of E2F is the transcriptional activation of cellular genes that encod proteins important for creating the S phase environment and allowing DNA replication to occur.
E6 targeted at the cell cycle check point controlled by p53 which is bound and degraded by E6.

3. Natural history of HPV infection
It generally takes 5 to 10 years for LSIL to progress to HSIL and another 10 to 20 years to invasive Ca. About 1/3 of HPV infection persisted, 1/10 progress to CIN and less than 1/100 ti Ca.

3. Histological Change of HPV infection:

A) Condyloma acuminata: Papillomatosis, acanthosis with hyperkeratosis, parakeratosis, presence of nuclear atypia and periunclear hals (koilocytosis).
B) Low grade squamous intraepithelial lesion (LSIL): Aberrant differentiation (mild dysplasia), perinuclear atypia (Koilocytes), absence of abnormal mitotic figure. Comparable to CIN Ⅰ.
C)High grade squamous intraepithelial lesion (HSIL): Plus the presence of abnormal mitotic figure. Comparable to CIN II,III and CIS.

*Flat cervical condyloma and low grade CIN have the same histology picture.
*Nuclear atycality: enlarged hyperchromatis smudged nuclei, empty or fragmenting nuclei.
*Abnormal mitotic figure: multinucleation, abnormal metaphase, bizarre forms,empty 
or fragmenting.

III. Perspective of HPV Infection Disease

Other Etiologic Factors of Cervical Carcinogenesis:
        Infection of HPVis essential but both insefficient and insufficient for cervical carcinogenesis. In thedevelopment of cancer, it usually takes sij or more “hit” on critical growth control genes (oncogenes or tumor suppresser genes). Infection of HPV leads to inactivation of p53 and Rb, but other etiologic factors are required for the other 4 or more “hits”. Smoking, estrogen and other chronic infections are proven risk factors. All are genotoxic which may lead to genomic instability (or microsatellite instability) and rapid accumulation of genetic hits. 

Application for High Risk Human Papillomavirus(HPV) Detection
Background:

■Evidence of HPV infection linked to pathogenesis of CIN 2& 3l 

●Cancer associated HPV’s are found in 95% of CIN 2 & 3 vs. 10% of normal women, yielding a relative risk estimate of 80:1l 
●Non-infected cervical keratinocytes are immortalized by oncogenic HPV infection.l 
●Histologic features of CIN 2 & 3 can be reproduced in vitro and iv vivo by oncogenic HPV infection of previously normal human keratinocytes. 
●Progressive potential of minor cervical atypia is influenced by HPV type. 

HPV Infection linked to progressin from CIN 2 to 3 to invasive cancer

●Cross sectional data show strong, consistent relationship between specific 
HPV types and both precursor and invasive disease.
●The HPV-immortalized human cells can eventually develop tumorigenic
(invasive) properties with long term culture.
●HPV Viral genome (especially E6 & E7) is continuously transcribed within cancer cells andcervix cancer derived cell lines.
●The E6 & E7 viral proteins bind tow cellular anti-oncogenes (p53 and pRb) that control cell growth rated.
●The HPV DNA is episomal in benign lesions but is integrated into cellular genome of most cancer cells. 

HPV is a Diagnostic Test supplementing a screening program

“Screening tests are relative simple procedures, designed to separate healthy persons from those who may have disease. They are not intended to be diagnostic but to identify patients who require a formal evaluation…….the use of HPV testing conforms to the basic philosophy of clarifying the nature and prognosis of any underlying disease” R. Reid, A. Lorincz New Generation of HPV Tests 1996.

Management of Equivocal Cytology results

AS a supplement or Triage for Atypical / ASCUS / AGUS Smears, either in conjunction with second smear or on its own (this way a test can be performed earlier than the 3-5 month delay necessary before a second smear can be taken, and referral based on the HPV shows better sensitivity than second Pap smear.

Patient is able to get information earlier than waiting for second Pap smear, which can relieve some anxiety and validate an earlier referral to colposcopy.Women could avoid a colposcopy and less colposcopies would be performed.(Pathology services are retained and increases market value before being referred to gynecological services) Subsequent screening cycles and management could be improved.
Screening Applications, Older women

Cytology in peri-and post mennopausal women is problematic; relative proportion of positive smears representing invasive cancer increases 17 times in 40 year olds vs 20 year olds; cytologic false negative ratea rise with age due to transformation zone recession into the cervical canal; falis positives rise in this age group becaouse of the confounding effects of estrogen deficiency.

HPV high risk positivity in older women is indicative of long term persistent infection, and higher risk for developing dysplasia.

Ease the concern of some clinics about the biannual not being sufficient as a screening cycle. They continue to recommend annual. HPV testing would identify those that would benefit from annual screens, as well as alternate technologies.

Risk Assessment

HPV High Risk Detection in Pap Smear negative women is predictive of an increased risk of subsequent detection of CIN, Cox Obstet & Gyn Clinics of Nth America 1996; Vol 23:811-851

Women over 30 could be assessed for theur HPV status as a means of determining risk for developing cervical disease.Test for the cause rather than wait for the effect.

Suggestive of closer scrutiny and more reqular Pap smears.
Validate the ejpense of additional Pap smears technology, Papnet/thinPrep/AutoCyte Detect Lesions before more extensive treatment would be needed.(No referral would be based on HPV positivity/ normal Cytology results.)

Management of Low-Grade Cervical Disease; Predicting Regression, aPersistence, or Progression of Untreated CIN1 

Most LSIL will regress (median value from # studies is 48% regress,and 12% progress),and trestment (intervenyion) should be based on presence of high risk HPV.Research has demonstrated that greater than 95% of HSIL and cancers contain oncogenic HPV, whereas very few non specific inflammatory/reparative changes will be colonized by such viruses. Alternative screening technologies are increasing the number of LSIL smears, if all were referred the additional vvisits will overwelm the colposcopic services and result in unnecessary intervention.

Treatment of CIN 1 or LSIL would be specifically indicated if patient was HPV high risk postive. A more conservative or “wait and see” approach could be adopted on the HPV negative patients.

Patients would have a treatment option. Gynaecologists would prioritize referrals based on HPV result, and could immediately treat evev where there was minimal aceto white presence. It situations where the colposcope was normal, the HPV result would support regular cytologic follow up.

Management of Equivocal HISTOLOGY

Postive HPV test would identify a subset of women who have either occult SIL missed at initial colposcopy or an emerging lesion that will manifest in the near future.

A negative or equivocal result is reported in approx 30% of patients who have directed biopsy for LSIL (Montz 1992). The decision to continue to triage or treat versus suspending work-up or returning patient to cytological follow up is difficult. A normal Coposcope and negative HPV result confer a negative predictive valug greater than 98%.Virus testing obviates any clinical concern over sighificant cyto-histologic discrepancy.

HPV could be used to manage biopsy equivocal patients, to determine follow up and cytology and/or colposcopic cycles. Reduce anxiety in proportion of patients.

Viral Load significant marker to managem and follow-up of LSIL Post Treatment

Measuring quantitative levels of HPV DNA may increase the predictive vaiue of HPV testing as a detection tool for HSIL.

Additional consideration when considering management options in HPV positive women. Assists in determining a conservative treatment approach or a more aggressive approach in CIN 1 and equivocals.

Assist in women determining what management option they should consider.
Detect the Presence of HPV as a follow up for Treatment;
Predicting Recurrence of Disease Post Treatment

The recurrence rate of CIN post treatment is usually around 10-15%.After treatment for HSIL, presence of HPV determines the screening and management options for the patient.

If subsequent to treatment Pap smears are negative, & HPV high risk negative, the women can return to normal screening.

Ability to give some long term prognosis to the patient, and a way of indicating whether there is further risk, or chances of re-occurrence of disease.In young women where repeated cervical ablation or may have severe consequences on fertility, HPV may be an objective method of determining patients at greatest risk.