I the name of God

HPV screening and genotyping

by molecular methods

Maryam sadat MirtalebiClinical and anatomical pathologist

Flowship of molecular pathology and cytogenetics

HPV

Human papillomavirus (HPV) genomes• Early genes: 50% of the genome and carries 6 ORFs, E1, E2, E4, E5, E6,

and E7• Late genes:40% of the genome, L1 and L2 encode the major and minor

viral capsid proteins used in the construction of new viruses.• Upstream regulatory region :long control region (LCR) (but also referred to as the noncoding region [NCR] or upstream regulatory region [URR]:covers about 10% of the genome and contains the origin of replication,multiple transcription binding sitesseparated by two polyadenylation (pA) sites (early pA and late pA).

Early genes:• E1 and E2: • initiation of viral DNA replication• regulation of early transcription.• E4 protein:• is expressed during productive infection • facilitate virus assembly • release of virions from infected cells. • The E5,E6, and E7 :• not universally present in all HPV types• function to modify the cellular environment to support completion of

virus replication.

Presence of the E5 gene• Associated with oncogenic potential• In high-risk HPV types :Interact with a variety of cellular targets, with subsequent effects that may contribute to: transformation of Epidermal growth factor receptorsOverexpression variety of proto-oncogenes Evasion of immune detection Inhibition of apoptosis

The major transforming activity of high-risk HPVs is due to the E6 and E7 genes, which interfere with regulators of the host cell cycle and control of transcription

Papillomavirus taxonomyThe present classification system was accepted in 2003, and there have been proposals for future amendments to harmonize the common naming usage in the scientific community with official groupings recognized by the International Committee on Taxonomy of Viruses (ICTV)

The current approach to papillomavirus taxonomy does not follow the classic rules for nomenclature, since there is not a practical culture or serological system.

Papillomavirus classification is based on the sequence of a 291-bp segment in a highly conserved region of the L1 ORF gene spanned by primers MY09 and MY11

The family Papillomaviridae

Alphapapillomaviruses BetapapillomavirusesGammapapillomavirusesMupapillomaviruses Nupapillomaviruses

HPV TYPES

• Strains within an HPV species are commonly described as “types” and are designated HPV followed by a number (e.g.,HPV16).

• New papillomavirus types are assigned when the completely characterized full-length genome has been deposited at the International HPV Reference Center at the Karolinska Institutet,Stockholm, Sweden, and the L1 ORF sequence is verified as differing by more than 10% (less than 90% similarity) from the closest known HPV type.

• Currently, 201 papillomavirus types isolated from humans (HPV1 to HPV205) have been completely sequenced and deposited.

Alphapapillomaviruses are categorized as high-risk or low-risk according to the likelihood that an infection by the HPV type can lead to the development of cancer. • In 2012, the International Agency for Research on Cancer (IARC) categorized HPVs

as:• group 1 (carcinogenic to humans) • group 2A (probably carcinogenic to humans)• group 2B (possibly carcinogenic to humans)

• The mucosal types can be divided into:• high-risk :16,18• low-risk (LR):6,11

HPV induced carcinogenesis

E6 mRNA, increased: Expression of p16 Decreased expression of cyclin D1Decreased expression p53Decreased expression Rb

Similar in cervical cancers among all group 1, 2A, and 2B carcinogens

HPV pathogenesis:• Alphapapillomavirus genus cause both mucosal and cutaneous lesions. • Beta-, gamma-, mu-, and nu-HPVs cause cutaneous lesions

HPV infection can follow either of two pathways:Productive :Supported by the normal basal epithelial stem cells found at the ectocervix

Transforming (nonproductive or abortive):Promoted in the specialized stem cell populations at the transformation zone

Productive infection

• The initial steps are the same regardless of the pathway.

• virions enter primitive basal epithelial cells by a complex binding proteins

• Virions initially bind to heparan sulfate proteoglycans on the surface of basal epithelial cells or basement membrane to cleavage site at the amino terminus of L2

If infection continues:A. The viral DNA is transported to the host cell nucleus in complex with the L2

minor capsid protein.B. An initial round of genome amplification occurs independent of the host cell

cycle, and copy numbers reach about 100 nuclear episomes per cell

C. The virus is maintained in low copy numbers in the nuclear episome phase, with minimal expression of early proteins E1 and E2, until the host keratinocyte begins to divide.

D. When the E2 protein reaches high levels, expression of the E6 and E7 genes is turned off and a read-through into the late region of the genome occurs, resulting in expression of HPV late genes L1 and L2

HPV can persist in the environment and remain infectious for at least several days without a host

• The highest risk for cancer development occurs as a result of prolonged persistent infection over many years.

• The nature of the replicative cycle of high-risk HPV types and a battery of immune evasion and subversion mechanisms allow the viral genome to be maintained at an almost constant copy number over many cell divisions

• In this process, the viral genomes replicate once along with host cellular DNA during the S phase of the cell cycle and are divided equally between the two resulting daughter cells

HPV integration

• occurs randomly at many sites in the human genome:genomicfragile sites • The break in the viral genomeOccurs in the E2 ORF causes deletion or disruption of the E2 gene retaining the E6 and E7 oncogenes and the LCR. Integration results:Downregulation of E2-mediated transcriptional

repressionOverexpression of the E6 and E7 oncogenes Activation of human telomerase.

The E6 and E7 proteins in high-risk HPV types• Regulation of the cell cycle, apoptosis,maintenance of chromosomal stability• inactivation of two host cell tumor suppressor proteins, p53 and

retinoblastoma proteins (pRb).• Binding of the viral E7 protein to pRb causes release of E2F and other proteins

that serve as signals for the cell cycle to progress.• The HPV E6 protein is a ubiquitin ligase and contributes to oncogenesis by

attaching ubiquitin molecules to p53,thereby making it inactive and subject to proteosomal degradation.

• E6 protein also activates telomerase, which synthesizes telomere repeat sequences and maintains a repeated cell cycle that continues to produce infected cells.

• The disruption of the normal p53 and pRb/E2F cell cycle regulatory mechanisms leads to the emergence of clonal cell populations that form a lesion.

Hallmarks of Cancer Analysis of HPV-Associated Cervical Carcinogenesis

Overexpression of p16 is a surrogate biomarker of HPV infection, the high risk types in particular which makes it useful in evaluating HPV-associated

squamous and glandular neoplasia of lower gynecologic tract

Infection with human papillomaviruses

HPV, one of the most common STIsWarts on cutaneous epithelium In the anogenital region can cause both genital warts and various forms of

cancer in men and women. Nearly 100% of cervical cancer are linked to a persistent infection by a high-risk

HPV (Human Papillomavirus)

HPV and Cervical Cancer Necessary precursor of both cervical cancer and associated precancerous

lesions, it is not a sufficient cause The majority of women with the infection will not progress to cancer Five percent of women infected with HPV who receive no health intervention

ultimately develop cervical cancerWoman’s lifetime risk of HPV infection at 75 percent. In 90% of cases, the body “clears” HPV with its own immunity 70 percent of those initially diagnosed, the infection is undetectable within 2

years. Proportion of women with HPV infection develops low-grade cervical lesions.

Most of these low-grade lesions regress spontaneously Approximately 15 percent progress to high-grade cervical lesions within two

years. High-grade cervical lesions have a strong malignant potential About one-third of high-grade lesions progress to cancer within ten years

Cervical Cancer Screening

• Cervical cytology (also called the Pap test or Pap smear)

Women aged 21–29 years should have a Pap test alone every 3years. HPV testing is not recommended.

Women aged 30–65 years should have a Pap test and an HPV test(co-testing) every 5 years (preferred). It also is acceptable to have a Pap test alone every 3 years.

• Human papillomavirus (HPV) Testing

Techniques for Detecting HPV

HPV cannot be cultured reliably in a laboratory setting; therefore, HPV diagnostics rely on molecular technologies that detect HPV DNA in cervical/vaginal samples.

Molecular techniques

1-Not amplified:nucleic acid probe tests2-Amplification :polymerase chain reaction (PCR): (1) target amplification: assay amplifies the target nucleic acids (2) signal amplification: signal generated from each probe is

increased by a compound-probe or branched-probe technology (3) probe amplification: probe molecule itself is amplified (for

example, ligase chain reaction).

Signal-amplified techniques for detecting HPV include• Hybrid capture• Branched DNA approachesThe most widely used technique is the hybrid capture technology

Non-amplified technologies:

Southern blot hybridization dot blot hybridization in situ hybridizationSeveral factors limit the incorporation of non-amplified techniques into large-scale screening programs:a. low sensitivityb. time-consumingc. require trained techniciansd. demand an array of laboratory reagents and equipment.

Current HPV tests

1. High risk HPV DNA tests without genotyping2. High risk HPV DNA tests with limited/partial

genotyping (genotypes 16 and 18)3. HPV DNA tests with full genotyping (Both low risk

and high risk genotypes)4. High risk HPV mRNA tests

* In situ hybridization-based HPV tests

Current HPV tests1. hr HPV DNA tests

Group of qualitative or semi-quantitative tests detecting hr HPV types without genotyping

• Hybrid Capture® 2 (HC2) HPV DNA test Evaluated in numerous randomized, controlled and cohort studies demonstrating the

clinical value of HPV testing in general

New HPV tests do not require extensive longitudinal clinical trials but should show equivalent(non-inferior) clinical characteristics as compared to HC2

• PCR-based consensus GP5+/6+ primers testcommercialized as EIA kit HPV GPHR by Diassay, The Netherlands)

• Cervista® HPV HR Test Signal amplification Invader chemistry

• care HPV Test (Qiagen, USA)

Simplified HC2 technology

Arbyn M. et al., Vaccine 2012;30(S5):F88-99Belinson JL. et al., Am J Clin Pathol 2011;135:790-5Poljak M., Kocjan BJ. Exp Rev Anti Infect Ther 2010;8:1139-62Qiao YL. et al., Lancet Oncol 2008;9:929-36Cuzick J. et al., Int J Cancer 2006;119:1095-1101

Current HPV tests:2. hr HPV DNA tests with limited/partial genotyping

Design of these assays is based on exceptionally high oncogenic potential of HPV16 and HPV18 as compared to other hr HPVs

• cobas® 4800 HPV test (Roche Diagnostics, Switzerland)Multiplex real-time PCRDetection for hrHPV and partial individual typing for HPV16 + HPV18

• Abbott RealTime High Risk HPV test (Abbott, USA)Multiplex real-time PCRDetection for hrHPV and partial individual typing for HPV16 + HPV18

• Cervista® HPV 16/18 Test (Hologic, USA)Reflex test for Cervista HPV HR + samples

Khan MJ. et al., J Natl Cancer Inst 2005;97:1072-9Wright Jr TC. et al., Am J Clin Pathol 2011;136:578-86Einstein MH. et al., Gynecol Oncol 2010;118:116-22Cuzick J. et al., J Med Virol 2010;82:1186-91

HPV DNA tests with full genotyping

• Group of HPV tests with detection and individual determination ofmultiple HPV types

• In contrast to previous groups of tests the clinical value of HPV full genotyping has not been definitely established

• Mostly used in research settings (natural history, transmission, pathogenesis, prevention)

• Analytical performance different (= higher analytical sensitivity) from the tests with clinically validated cut-offs

• Role in the surveillance of vaccine effectiveness on population level

Current HPV tests: 3. HPV DNA tests with full genotyping

• Reverse hybridizationMost frequently used HPV DNA full genotyping tests

Linear Array® HPV Genotyping test

HPV Direct Flow Chip (Master Diagnostica)CE-IVDBased on PCR amplification and reverse dot blot hybridization

• Microarray-based testsPapilloCheck® HPV-Screening Test/PapilloCheck High-risk Test (Greiner Bio-One, Germany)Clart® HPV2-Papillomavirus Clinical Arrays (Genomica, Spain)

• Microsphere beads-based testsUltrasensitive methods

Gravitt PE. et al., Vaccine 2008;26(S10):K42-52Hesselink AT. et al., J Clin Microbiol 2010;48:797-801Schmitt M. et al., J Clin Microbiol 2010;48:143-9

Current HPV tests4. hr HPV mRNA tests

Most relevant transcripts for diagnostic purposes are those encoding viral oncoproteins E6 and E7

Reverse-transcriptase PCR of nucleic acid sequence-based amplification (NASBA)

• APTIMA ® HPV Assay

Targets E6/E7 mRNA of 12 hrHPV + HPV66 + HPV68No discrimination among 14 targeted HPV typesNo cellularity controlNot only HPV mRNA but also HPV dsDNA detectionIn traige settings: = sensitivity but ↑ specificity than HC2 for detecting cervical

precancer

lesions• PreTect HPV-Proofer

↓ sensitivity but ↑↑ specificity than DNA-based tests for CIN2+•

Detection of E6/E7 mRNA of the 5 most frequent hrHPV types = HPV16, HPV18, HPV31,

HPV33, HPV45•

Not only HPV mRNA but also HPV dsDNA detection

Current HPV tests* In situ hybridization-based HPV tests

• The only molecular method allowing topographical relation of HPV detection to pathological lesions.

Note: Once samples are taken, they can be stored at roomtemperature for two weeksat 4°C for one additional weekat -20°C for up to three months.

ASCCP guidelinesAmerican society for colposcopy and cervical pathology

FUTURE CHALLENGES

• Current cervical cancer screening strategies using cytology with or without HPV testing have been effective

Future: focus on improving the efficiency effectiveness of screening better management of patientspreventing increased costs• The importance of HPV testing has been recognized, and its role in cervical

screening is shifting from cytology alone to cytology plus HPV cotesting and now to the recognition of a new screening paradigm in which HPV testing can function alone as the primary screening test.

• Beyond cytologic examination and testing for the presence of high-risk HPV, there is a continuing need to find viral and/or host markers of disease progression that will help distinguish clinically insignificant, selflimited HPV infections from precancerous infections.

Conclusion• HPV has clearly been shown to be the cause of most cervical cancers. Given

that, interest is growing worldwide in the potential for use of HPV diagnostics in cervical cancer prevention programs, both as an adjunct to cytological screening and in primary screening for cervical dysplasia.

• In developing countries, the currently available tests likely are too expensive and technologically demanding for widespread use, even though research in several countries has demonstrated their potential for identifying high-grade dysplasia in older women (age 35 and older).

• Developing country programs interested in incorporating HPV DNA testing into cervical cancer prevention activities may have to wait for the development of HPV tests that are less expensive than existing options, and easier to use in nonlaboratory settings.

• At the same time, screening programs based on HPV testing must be carefully designed to ensure that the tests are used in a way to maximize their effectiveness in detecting high-grade dysplasia in women at highest risk of developing cervical cancer.

Thanks for your attention

I the name of God��HPV screening and genotyping�by molecular methodsSlide Number 2Human papillomavirus (HPV) genomesSlide Number 4Early genes:Presence of the E5 geneSlide Number 7Papillomavirus taxonomyThe family PapillomaviridaeHPV TYPESSlide Number 11Slide Number 12HPV induced carcinogenesisSlide Number 14HPV pathogenesis:Productive infectionIf infection continues:Slide Number 18Slide Number 19HPV integrationThe E6 and E7 proteins in high-risk HPV typesSlide Number 22Hallmarks of Cancer Analysis of HPV-Associated Cervical CarcinogenesisSlide Number 24Infection with human papillomavirusesSlide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Cervical Cancer Screening�Slide Number 33Molecular techniques�Slide Number 35Non-amplified technologies:Current HPV tests�����Current HPV tests� 1. hr HPV DNA tests�������Current HPV tests:� 2. hr HPV DNA tests with limited/partial genotyping��HPV DNA tests with full genotyping �Current HPV tests: � 3. HPV DNA tests with full genotyping ��Current HPV tests�4. hr HPV mRNA tests����Current HPV tests� * In situ hybridization-based HPV tests��Slide Number 44Slide Number 45ASCCP guidelinesSlide Number 47Slide Number 48Slide Number 49Slide Number 50Slide Number 51Slide Number 52FUTURE CHALLENGES�ConclusionSlide Number 55