This document provides information about human papillomavirus (HPV) including: - There are over 100 types of HPV that can infect humans. High risk types like HPV 16 and 18 can cause cancers while low risk types cause genital warts. - HPV is transmitted through skin to skin contact and infects mucosal surfaces. Most infections clear on their own but persistent infections can lead to cancers. - HPV is associated with cervical, anal, penile, and oropharyngeal cancers. It is also associated with genital warts. - The viral life cycle and genes involved in pathogenesis are described. Integration of viral DNA into host DNA is important for cancer development. - Screening methods

1. Presentor : Dr.
REEMA AGRAWAL
Moderator: Dr.
SURABHI TYAGI

3. • FAMILY : PAPOVAVIRIDAE
• GENUS: PAPILLOMAVIRUS
• Infects the epidermis and mucous membranes
of humans.
• Highly species specific

4. • HPV infections occur on skin and mucous
membranes, in the conjunctiva, oral cavity,
larynx, tracheo-bronchial tree, esophagus,
bladder, anus, and genital tract of both sexes.
• Approximately 140 HPV types have been
identified.

5. • More than 80 HPV genotypes have been
identified.
• 16,18,31,33,35,39,45,51,52,56,58,59,6
6,and 68
14 High risk
(oncogenic
types):
• 6, 11, 42, 43, 44…..
Low risk
(nononcogenic
types):

6. • HPV- DNA prevalence in normal women varies from 7.63%
in Asia which is among the lowest to 23.41% in Africa which
is the highest.
J Natl Cancer Inst 1995;87:796-802.
• The peak
prevalence is
seen in women
15 to 25 years
of age and then
declines with
age.

7. According to the Centers for Disease Control
(CDC),In U.S.-
• 20 million people are infected.
• HPV prevalence is estimated to range from 15-
25% in U.S. women.
• By the age of 50, more than 80% of American
women will have contracted at least one strain
of genital HPV.
• Around 6.2 million new infections occur each
year.

8. Studies in India show:
1. 98% HPV positivity in invasive cancer cases v/s 20%
in normal healthy controls;
2. HPV 16 as the predominant (53-62%) type while the
frequency of HPV type 18, is very low (13-15%);
3. HPV 16 is also more frequent than HPV 18 in
cervical adenocarcinomas; (worldwide HPV 18 is
more frequent in adenocarcinomas)

9. 4. HPV infections are at least two-times more
frequent in pregnant women than in non-pregnant
women.
5. Sexual intercourse before 18 years of age has
been found to increase the risk of cervical cancer
by 22 fold.
CURRENT SCIENCE, VOL. 78, NO. 1, 2000), (Lancet, 89)

10. HPV 16, 18:
• Cervical Cancer.
• High grade cervical abnormalities.
• Low grade cervical abnormalities.
• Anal cancer.
• Vulvar/ Vaginal/ Penile Cancer.
• Head and neck cancer.
HPV 6,11:
• Low grade cervical abnormalities.
• Genital warts.
• Recurrent respiratory papillomatosis (RRP).

11. • Cervical cancer 95-98%
• vaginal carcinoma 90%
• Vulval carcinoma 30-35%
• Anal cancer 80%
• Penile cancer 50%
• Head and neck cancers <12%
( Oral SCC, tonsillar carcinoma)
Others
• Lung carcinoma
• Gastrointestinal carcinomas

12.  The papillomaviruses are small non-enveloped
icosahedral viruses (55nm)
 Seventy-two capsomeres surround the genome.
 A major and minor capsid protein comprises the outer
protein coat of the virus
 have a circular double stranded DNA genome

13.  HPV have a naked icosahedral capsid
 The capsid consists of 72 pentamers that are either
pentavelent or hexavalent
 Each pentamers is composed of five major capsid
proteins
 L1 protein has a molecular weight of about 55 kd and
makes up about 80% of the total viral protein.

14.  The L2 protein is the minor capsid protein, which has
a molecular weight of 70 kd.
 Both the L1 and L2 proteins are made in the
cytoplasm of the infected cell during the late phase of
infection. They are then transported to the nucleus
where they are assembled into infectious particles.
 minor capsid, L2, is located internal to the L1 shell.

15. • The viral genomic DNA is packaged within the
L1/L2 capsid as a mini chromosome
• The recombinant L1 capsid alone can assemble into
the virus-like particle structure in vivo and in vitro
• L1 protein contains all the information needed for
the particle assembly.

16.  Genomic organization of the different types of HPV are
same
 HPV viral DNA can be divided into 3 regions
URR(upstream regulatory region):
• Non coding region of the viral genome
• Important in regulating viral replication and transcription
of downstream sequences in the early region

17. • Contains binding sites for various transcription factors
including activator protien1(AP1)
Early(E) Region:
• consist of six different open reading frames (ORFs) - E1, E2,
E4, E5, E6 & E7.
• They encode for proteins required for viral replication and
maintenance of a high viral copy number in infected cells

18. Late (L) Region:
 Downstream of the early region
 consists of 2 ORFs (open reading frames) - LI and
L2.
 L1 and L2 regions are expressed during active
viral production and shedding as a late event in the
viral life cycle

19. Gene Function
E1 Initiation of DNA replication
E2 Transcriptional regulation/DNA
replication
E3 ?
E4 Disrupts cytoskeleton?
E5 Transforming protein, interacts with
growth factor receptors
E6 Transforming protein, binds to p53,
leading to degradation
E7 Transforming protein, binds to pRB
E8 ?
L1 Major capsid protein
L2 Minor capsid protein

21. • Initial site of infection is basal cells of immature squamous
epithelium
• Two types of infection
Latent infection
 Maintenance of viral infection without production of
infectious virus
 Viral DNA remain in the form of episome

22. • Replication of episomal DNA tightly coupled to the
replication of the epithelial cells
• Replicate with host cell’s DNA
• Complete cytopathic effect of HPV infection are not
present
• No morphological abnormalities

23. Productive infection
• DNA replication occurs independently of host chromosomal
DNA synthesis
• Produces large amount of viral DNA
• Predominantly in intermediate and superficial cell layers
• Produces large amount of virions
• Cytopathic effects of HPV can be detected cytologically
and histologically
• Cytoplasmic vacuolization, nuclear atypia and
multinucleation seen

25. Precursor lesions for cervical cancer

27. HPVS: NATURAL HISTORY
WITHIN 1 YEAR
INITIAL HPV
INFECTION
CLEARED HPV INFECTION

28. HPVS: NATURAL HISTORY
WITHIN 1 YEAR 1-5 YEARS1-3YEARS
INITIAL HPV
INFECTION
PERSISTENT
INFECTION
CIN 1
CIN
2/3
CLEARED HPV INFECTION

29. HPVS: NATURAL HISTORY
WITHIN 1 YEAR 1-5 YEARS1-3YEARS UPTO DECADES
INITIAL HPV
INFECTION
PERSISTENT
INFECTION
CIN 1
CIN
2/3
CERVICAL
CANCER
CLEARED HPV INFECTION

30. Anogenital infections :
• by sexual contact.
• Infection is facilitated by the presence of macerated or
abraded epithelial surfaces.
Occasionally transmitted:
• perinatally to infants during delivery.
• digitally from one epithelial site to another site.
• by oro-genital contact to oral site.
• by contact with contaminated material.

31. HPV CLEARANCE
• Approximately 70% of new infections clear within one
year, 91% within 2 years.
• Most clearance in first 6 months.
HPV PERSISTENCE
• Infection detected at more than one visit.
(usually 4-6 months apart)
• Most important predictor of high grade cervical cancer
precursors.

32. CONSISTENT-
• Sexual behavior:
-younger age of sexual initiation.
-no. of sex partners.
-partner’s sex partners.
• Immune status.
• Age (<25 years).
• History STD.
LESS CONSISTENT-
• Smoking.
• Diet.
• Hormonal Contraceptives.
• Inconsistent Condom use.

33. HPV INFECTION IN MEN
• Equally prevalent among males as females.
• Natural history data is limited.
RISK FACTORS FOR MEN
• Consistent:
-Sexual behavior.
-Immune status.
-Lack of circumcision.
• Less Consistent:
-History STD.
-Inconsistent Condom use.

35. • Second most common malignancy among women
worldwide and most common in India.
• Most cancers occur in the transformation zone of the
cervix and 85% are squamous cell carcinomas.
• At least 99.9% of cervical cancers contain HPV DNA.
• Worldwide HPV 16 is the most common type found in
cervical cancers.

36. • E6 and E7 forms the principal transforming genes of
HPV
• Expression of E6 and E7 ORF’s from high risk
HPV’s causes the cells to become completely
transformed

37. • In low grade SIL (CIN1) and in most high grade
SIL(CIN2,3), HPV is episomal and E2 ORF is intact
• In most cancers HPV DNA is integrated into the
cellular DNA
• Integration occur into the E1/E2 region, leading to
disruption and inactivation of the ORF’s
• resulting in over expression of E6 and E7

38. • E6 protein binds and causes the rapid degradation of
the p53 protein, which is an important regulator of
cellular growth and differentiation
• E7 protein deregulate cell growth by binding
cyclinA,p107 and p105 RB, which regulate the
progression of cells from G1 to S phase
• Resulting in a loss of growth control

39. The E6 gene p53 (tumour suppressor gene)
The E7 gene retinoblastoma gene product (pRb)
E2 gene product promote a mitotic block
Dysregulation of the cell cycle cells with genomic defects
to enter the S-phase.
Promote chromosomal instability, Induce cell growth
and immortalize cells.
Oncogenesis
Pathogenesis of cervical cancer

41. Mucosal/genital
(~40 types)
Nonmucosal/cutaneous
(~60 types)
• Subclinical infection
• Exophytic condyloma
• Flat condyloma
• Bowenoid papulosis
• Cervical cancer
• Vulvar cancer
• Penile cancer
• Recurrent Respiratory
papillomatosis
• Oral and tonsillar cancer
• Nongenital skin warts
• Epidermodysplasia
Verruciformis(EV)
• Nonmelanoma Skin Cancer

42. HPV types Most common clinical
lesion
Less frequent lesion Potential Oncogenicity
1 Deep planter/palmer
warts
Common warts
2,4,27,29 Common warts Palmar
,planter.mosaic, oral
and anogenital warts
3,10,28,49 Flat warts Flat warts in EV HPV-10 rare in cervical and
vulvar carcinomas
7 Butcher’s warts
13,32 Oral focal epithelial
hyperplasia
5,8,9,12,14,15,17,1
9-26,36,47,50
EV, warts in
immunosupression
Normal skin (?) HPV-5,-8,-9 isolated from
SCCs
6,11 Anogenital warts,
cervical condyloma
Bowenoid papulosis,
common warts,
respiratory
papillomatosis,
common warts
Buschke- Lowenstein tumor,
rare in penile, vulvar cervical,
and other urogenital tumors;
“low risk”
16,18,31,33-35,39-
40,51-60
Cervical condyloma,
anogenital warts;
bowenoid papulosis
Common warts Genital and cervical
dysplasias and carcinomas,
rare in cutaneous SCC: “High
risk”
>100 different types of HPV virus Greater than 10% difference in nucleotide
homology within the L1 gene

44. Clinical inspection
Biopsy if indicated
Acetowhite test in
flat condyloma

45. • Virus cannot be cultured in vitro.
• Serology:
-Low sensitivity and specificity (because of
lack of good source of viral antigens

47. 1. Computerised call and recall from a population
registry
2. Invitations and reminders
3. Sample taking by trained sample takers
4. Quality assured cytology reporting
5. Refferal to colposcopy
6. Integration into an organised, monitored quality
assured programme.

48. “What distinguishes cancer
screening programme from
other forms of care is the
presence of quality assurance
as part of the national
programme.”

49. • Primary cervical cancer prevention programme is
based on cytological screening.
• Drawbacks: Technical limitations, low sensitivity,
inter-screener variations and diagnostic errors.
• But, the specificity of the cytology test is very good.
• HPV-DNA test cannot fully replace the Pap-test.

53.  The English screening programme currently uses
cytology as the primary screening modality with an
HPV test to triage borderline and low grade
abnormalities. This is a new policy rolled out during
2012, and replaces cytology screening only.
 Screening intervals in England are 3 yearly from age
25 to 49 and 5 yearly from age 50 to 64.

54. • Co-testing leads to earlier diagnosis of CIN 3+
and cancer.
• Incorporating HPV finds more
Adenocarcinoma in situ than cytology alone.
• Negative cytology plus negative HPV allows
spacing screening beyond every three years.

57. • Routine screening in women <age 30 (except
ASCUS triage).
• Women =/>24 years with ASC-H, LSIL,
HSIL, AGC( except post menopausal LSIL
reflex HPV testing).
• Women considering HPV vaccination.
• Routine sexually transmitted disease
screening.
• As part of sexual assault work up.

58. Finding carcinogenic HPV types
doesnot provide a diagnosis of
CIN 3 or cancer
It identifies a group of women in
whom CIN3+ is more likely.

59. 1. Triage of low grade cytology abnormalities:
allows women who are HPV negative to
revert to routine recall as their risk of having
significant disease is extremely low, and
allows selection of the higher risk women for
immediate colposcopy.
2. Test of cure- checking complete reversion to
negative after treatment of CIN.
3. As a primary screening test

60.  The strength of HPV primary screening is its
sensitivity, and this can be achieved more easily than
with a cytology-based programme.
 The limitation of HPV testing is its specificity.
Clinically, a positive HPV test result can only
prioritise for further investigation, whereas a negative
HPV result is very useful and can provide for further
reassurance.

61. 1. Digene hybrid capture 2 HR HPV DNA Test
2. Hologic Cervista
3. Roche Cobas
4. Abbott Real Time
5. Genprobe Aptima
“All these tests uses ThinPrep or SurePath LBC samples
for the detection of HPV”

62. • is a nucleic acid hybridization assay with signal
amplification that utilizes microplate
chemiluminescent detection technique.
• 13 HR types targeted
– Cross-hybridization: HR: 66; LR: 8, 9, 43, 45, 47
• No extraction
• No target amplification
• No internal control

64. 1.Target DNA
hybridize with a
specific HPV
RNA probe
cocktail.
2.RNA:DNA
hybrids are
captured
onto the
surface of a
microplate
well coated
with
antibodies
specific for
RNA:DNA
hybrids
3.Hybrids are then
reacted with alkaline
phosphatase
conjugated antibodies
specific for the
RNA:DNA hybrids,
4.As the substrate is cleaved by the
bound alkaline phosphatase, light is
emitted that is measured as relative
light units (RLUs) on a luminometer.
The intensity of the light emitted
denotes the presence or absence of
target DNA in the specimen

65. • An RLU measurement equal to or greater than the Cutoff Value (CO)
indicates the presence of high-risk HPV DNA sequences in the specimen.
An RLU measurement less than the Cutoff Value indicates the absence of
the specific high-risk HPV DNA sequences tested or HPV DNA levels
below the detection limit of the assay.
• A cut off of 2 RLU is used in England.
Limitations of the test:
• Only a semi-quantitative test (RLU scores are correlated with CIN2 and not with
HR HPV).
• A small amount of cross-hybridization between HPV types 6 and 42 (low-risk HPV
types) and the High-Risk HPV Probe exists.
• Specimens with high levels (4 ng/ml or higher) of HPV 6 or HPV 42 DNA may be
positive.

66. • The Hologic Cervista test is an in vitro diagnostic test for the
qualitative detection of DNA from Human Papilloma virus
(HPV) Type16 and Type 18 in cervical specimens.
• Hologic Cervista test uses the Invader®chemistry, a signal
amplification method for detection of specific nucleic acid
sequences. This method uses two types of isothermal
reactions: a primary reaction that occurs on the targeted DNA
sequence and a secondary reaction that produces a fluorescent
signal.

67. • DNA extraction method
• Probes for 14 HR HPV types- 16, 18, 31, 33,
35, 39, 45, 51, 52, 56, 58, 59, 66, 68
• No target amplification
• HPV 16 and 18 typing can also be performed
as a reflex using genotype-specific probes.
• Internal control used is histone 2.

68.  In the primary reaction, the
probes cycle rapidly on and off the
target. Each time an intact probe
molecule binds to the specific
target in the presence of the
Invader oligo, the overlapping
substrate is formed and cleavage
occurs. The number of flaps
released is relative to the amount of
target in the sample, allowing for
quantitative detection of genes,
chromosomes or infectious agents

69.  In the first reaction, two oligonucleotides, a probe and an
Invader oligo, anneal to a specific DNA target sequence to
generate a one-base overlapping structure if the desired
sequence is present. The one-base overlapping structure is
created with the probe and the Invader oligo on the target.
Proprietary Cleavase® enzymes specifically cleave the
overlapping primary probes, releasing the 5' flaps plus one
nucleotide

70.  Secondary, Simultaneous
Reaction
Cleaved flaps from the primary
Invader reaction combine with a
fluorescence resonance energy
transfer (FRET) probe in a
secondary, simultaneous
overlapping cleavage reaction,
generating a fluorescent signal.
The combination of two different
flap sequences, FRET oligos, and
fluorophores allows for single-
well biplex reactions to occur.

71.  Final Results
Each released 5' flap from the primary reaction cycles
on and off the FRET probes, enabling the secondary
reaction to further amplify the target-specific signal.
The two simultaneous reactions typically produce a
1-10 million-fold signal amplification during a 4-hour
reaction.

72.  These two test are qualitative in vitro
polymerase chain reaction (PCR) assay that
utilizes homogeneous target amplification
and detection technology for the detection of
high risk human papillomavirus (HPV) DNA
in cervical cells collected in liquid cytology
media.
 Both are intended to detect 14 high risk HPV
genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58, 59, 66, 68 and to partially genotype
16, 18 from other 12
high risk genotype
Cobas 4800 system

73. • Simultaneously detects 14 high-risk HPV types and
provides specific genotyping information for HPV
Type 16 and 18.
• ß-globin from cellular input is used as an internal
control to assess specimen quality and identify
specimens containing factors that inhibit the
amplification process.

74. Specimen collection
 Specimens are limited to cervical cells
collected in cobas® PCR Cell
Collection Media, PreservCyt®
Solution and SurePath® Preservative
Fluid.
Abbott m2000 Real
Time PCR system

75. • The APTIMA HPV Assay is an in vitro nucleic acid
amplification test for the qualitative detection of E6/E7 viral
messenger RNA (mRNA) from 14 high-risk types of human
papillomavirus (HPV) in cervical specimens.
• The APTIMA HPV Assay does not discriminate between the
14 high-risk types
• The assay is used with the TIGRIS DTS System or the
PANTHER System.

76. • Specimen collection
Cervical specimens in ThinPrep Pap Test vials containing
PreservCyt Solution and collected with broom-type or cyto
brush/spatula collection devices may be tested with the APTIMA
HPV Assay.
Principles of the Procedure
The APTIMA HPV Assay involves three main steps, which
take place in a single tube:
1. Target capture
2. Target amplification by Transcription-Mediated Amplification
(TMA);and
3. Detection of the amplification products (amplicon) by the
Hybridization Protection Assay (HPA).

77. • When the APTIMA HPV Assay is performed, the target
mRNA is isolated from the specimen by use of capture
oligomers that are linked to magnetic microparticles
• During the hybridization step, the sequence-specific regions
of the capture oligomers bind to specific regions of the HPV
mRNA target molecule. The capture oligomer:target complex
is then captured out of solution by decreasing the temperature
of the to room temperature.
• The microparticles, including the captured HPV mRNA target
molecules bound to them, are pulled to the side of the reaction
tube using magnet and the supernatant is aspirated.

78. • After target capture is complete, the HPV mRNA is amplified
using TMA, which is a transcription based nucleic acid
amplification method that utilizes two enzymes, MMLV
reverse transcriptase and T7 RNA polymerase.
• The reverse transcriptase is used to generate a DNA copy of
the target mRNA sequence containing a promoter sequence for
T7 RNA polymerase. T7 RNA polymerase produces multiple
copies of RNA amplicon from the DNA copy template.

79. • Detection of the amplicon is achieved by HPA using
single-stranded nucleic acid probes with
chemiluminescent labels that are complementary to
the amplicon.
• The labeled nucleic acid probes hybridize specifically
to the amplicon. The Selection Reagent differentiates
between hybridized and unhybridized probes by
inactivating the label on the unhybridized probes.

80. • During the detection step, light emitted from the
labelled RNA:DNA hybrids is measured as photon
signals called Relative Light Units (RLU) in a
luminometer. Final assay results are interpreted based
on the analyte signal-to-cutoff (S/CO).
• IC(Internal Control) is added to each reaction via
the Target Capture Reagent. The IC monitors the
target capture, amplification, and detection steps of
the assay.

81. Test Method Targets
PreTect
Poofer(Norchip)
NASBA Five types
16,18,31,33,45
NucliSENS EasyQ HPV
v1(bioMerieux)
NASBA 16,18,31,33,45
HPV OncoTect E6/E7
mRNA (incellDx)
Flow cytometry Transforming cells
E6/E7 mRNA in each
cell

82.  In the English screening programme, a
comprehensive evaluation was performed comparing
these five tests with differnt LBC samples in a triage
setting.
 Results were consistent.
 Genprobe aptima showed a higher specificity, but the
differences were not great.

83. 1. P16, a cell cycle protein which is overexpressed
when E7 binds to p53, can be detected by
immunohistochemistry.
2. Ki67 may improve the specificity of a HPV + and
cytology low grade sample.
3. ProExc , a composite marker comprising
identification of mini chromosome
maintenance(MCM) and
4. Topoisomerase IIα (TOP2A), a marker of cell
proliferation.

85. • Two prophylactic HPV vaccines have been developed.
• They are based on the recombinant expression of the L1 major
capsid protein and subsequent self-assembly into virus like
particles (VLPs) that resemble the outer shell of the virus.
• VLPs contain no DNA and are not live/attenuated viruses.
Quadrivalent HPV vaccine. (Gardasil, Merck & Co.,
Inc., Whitehouse Station, NJ)
Bivalent HPV vaccine. (Cervarix, GlaxoSmithKline,
Middlesex, UK)

86.  HPV-16,18 (Cervarix, GlaxoSmithKline)
◦ Designed to prevent cervical cancer, other
malignancies.
 HPV-6,11,16,18 (Gardasil, Merck)
◦ Designed to prevent cervical cancer and other
malignancies, genital warts, RRP.
Efficacy :
The prophylactic vaccines for HPV have been demonstrated to
prevent persistent HPV 16 and 18 infections and HPV 16– and 18–
related CIN2/3 in various studies.

87. • Gardasil: FDA approved
–Indications
Prevention of cervical cancer and genital warts
caused by HPV 6, 11, 16, and 18 as well as
precancerous lesions (CIN, VIN, VaIN) in
girls and women 9-26 years of age
–Given as three IM injections in upper arm over
6 months (0, 2, 6 months)

88. • Routine HPV vaccination is recommended for females
aged 11 to 12 years.
• Females as young as 9 years may receive HPV
vaccination.
• HPV vaccination is also recommended for females 13
through 18 years of age to catch up missed vaccine or
complete the vaccination series.
• Screening for CIN and cancer should continue in both
vaccinated and unvaccinated women according to
current ACS early-detection guidelines.