This is a presentation about human papilloma virus that I delivered to MLT students last year.

1. Human Papilloma virus:
An Introduction to Oncogenesis
and Molecular Diagnosis
Presented by:
Bassim Zantout CT (ASCP), MBA

2. Human Papilloma Virus & Cervical
Cancer
 The causal role of HPV in
cervical cancer has been
documented beyond a
reasonable doubt.
 This role
has been furnished
through a long
history of research as
well as major
epidemiologic studies
which were supported
at later stages by
molecular technology.
 Human Papilloma Virus
has been Proposed as
the “Necessary Cause”
of Cervical Cancer.
 Evidence has been accumulating on the
role of HPV in other human cancers.

3. Human Papilloma Virus: A Brief
History
 A couple of millennia ago cervical
cancer was first described and was
considered as an incurable disease.
 A couple of millennia later later
around mid 19th century an
Italian surgeon (Rigoni Stern)
made the observation that
cervical caner was rare among
nuns.
 In the 1950s and the 1960s
Potential link between a viral
infection and cervical cancer was
first noticed by scientists who
were looking for clues about
what triggers cervical
cancer.
 An interesting observation was
also later made that cervical
cancer was more likely to occur
in women who start their sexual
activity at an early age
and who have multiple sexual
partners.
 However, those
researchers did not have
a definitive causative
factor and proposed
over the years a number
of infectious agents:
 Syphilis
 Gonorrhoea
 Herpes simplex virus type 2

4. HPV: A Brief History (cont’d)
 These findings piqued the interest
of a German virologist by the
name Harold zur Hausen
 Zur Hausen was well
aware of reports about
women with genital
warts who later developed
cervical cancer.
 Zur Hausen also knew
about an American
researcher by the
name Richard Shope
from the 1930s whose work
showed that an infection with
a type of HPV causes warts
that might progress to cancer
in infected rabbits.
 Zur Hausen began his quest
looking for viruses in human
genital warts.

5. HPV: A Brief History (cont’d)
 Zur Hausen’s research
and investigation lead
to the discovery of
HPV 16 which he
found in half of cervical
cancer cases and HPV 18
which he found in one in
five cervical cancer
cases.
 These discoveries earned zur
Hausen the Nobel Prize in
2008.

6. Establishing the
Strength of Association
HPV and Cervical Cancer

7. IARC Multicenter Study
 IARC multicenter study is a case-
control study on invasive cervical
cancer that encompassed nine
countries: Brazil, Morocco, the
Philippines, Paraguay, Thailand, Peru,
Mali, Spain, and Colombia.
 The study utilized PCR HPV DNA
testing System for the detection of
HPV infections.
 The study
reported the Odds
Ratio (OR)

8. Evidence-Based Medicine

9. Odds Ratio (OR) and Its Interpretation
 Its is a statistical tool that
is used in case-control
studies.
 OR compares the odds of
exposure in cases (having
disease) to the odds of
exposure in controls (no
disease).
 OR is used to determine
whether an exposure is
related to disease
outcome.
Interpretation
 OR=1
 Exposure does not affect disease
outcome or odds of disease.
 OR>1
 Exposure is associated with
higher odds of the disease.
(Risk Factor)
 OR<1
 Exposure is associated with
lower odds of disease.
(Protective)

10. Example
90 10
10 90
Cervical Ca No Disease
Exposed
Not Exposed
OR= 90*90/10*10
=8100/100
=81

11.  The prevalence of the four most common HPV types and their
ORs among 1545 cases with single infection were as follows:
IARC Multicenter Study (cont’d)

12. IARC Multicenter Study (cont’d)
 Other less common HPV types
showing equally high ORs
were identified
33 77.6
35 34.8
51 42.7
52 145.7
58 78.9
59 347.3
 These findings indicate
that in addition to HPV
16, 18 , 45 & 31HPV types
33,35,45,51,52,58,59 are
also considered as human
carcinogens

13. International Biological Study on
Cervical Cancer (IBSCC)
 The aim of the study was to determine the association
between HPV infection and cervical cancer.
 > 1000 specimens from patients with invasive cancer were
collected and stored in 32 hospitals in 22 countries.
 PCR technique was utilized to detect HPV DNA
Results
HPV was detected in 93 % of tumors.
HPV 16 dominated in 51% of
squamous cancers.
HPV 18 dominated in 56% and 39% of adenocarcinomas and
adenosquamous carcinomas respectively.

14. Proliferation in The Publications on HPV and
Cervical Cancer in the 80s and 90s
 The 1980s generated a rapidly increasing number of
publications on HPV DNA prevalence in cervical cancer.
 The 1990s produced key results of epidemiological studies:
case studies, cohort studies, case-control studies.
 The late 1990s showed an increasing number of publications
on the clinical use of HPV DNA testing and triage.

15. HPV Causes Cervical Cancer
 Today the scientific
and medical
communities can
safely say that HPV is
the Necessary
Causative agent of
human cervical
cancer.

16. Implications of HPV As
“Necessary Cause”
 High risk women can now be sharply
redefined as the group of persistent
HPV carriers.
 This represents a substantial
progress from the previous
version of high risk group that
identified women by their
exposure to a constellation of
ill-defined factors:
 Low socioeconomic
status.
 Multiple sexual partners.
 Use of oral contraceptives.
 History of STDs.
 Smoking.

17. Human Papilloma Virus Genome
Structure
 Papilloma virus is a small
non-enveloped virus with
icosahedral capsids that
contain circular double
stranded DNA.
 The genome is divided into
three major portions: (E)
&(L) regions and Long
control region (LCR)
 Variations in carcinogenic
potential among HPVs
are principally governed by
the E6 and E7 oncoproteins.
 E6 and E7 proteins are capable of
interacting with and altering, or
destroying, key cell cycle regulatory
molecules

18. Icosahedral Structure

19. HPV Genome
Linearized Version of HPV Genome E6 and E7 oncoproteins
form complexes with
tumor suppressors and can
lead to host cell
transformation (cellular
immortalization) genomic
instability and progression
to cervical cancer

20. Understanding
HPV Oncogenesis

21. HPV Oncogenesis
 Normal cell cycle and cell cycle
checkpoints.
 p53
 pRb
 Cyclins and cyclin-dependent
kinases (ckdases)
 Telomeres and Telomerases.

22. Normal Cell Cycle

23. Cell Cycle Checkpoints

24. p53 Gene
 Tumor suppressor gene.
 p53 protein (product of gene)
binds to DNA which in turn
stimulates another gene to
produce p21 protein.
 p21 reacts with cell-division
stimulating protein (cdk2).
 P21/cdk2 complex inhibits the cell from
passing into the next stage of cell
division
(S phase).

25. p53 Role as Tumor Suppressor
DNA
damage
Triggers
expression of p53
Increases p53
level
Prevent
cells from
entering S
phase
Arrest
cell
cycle
Allow
time for
DNA to
repair
DNA
repaird
P53
degrades
Cell
cycle
resumes
DNA not
repaired
Permanent
arrest
(Senescence)
Apoptosis

26. Cyclins
 Family of regulatory
proteins that control the
progression of the cell
cycle.
 Cyclins activate cyclin-
dependent kinases
(Cdkases), which control
cell cycle through
phosphorylation.

27. Cyclin Expression in the Different Phases
of The Cell Cycle

28. Retinoblastoma Protein (pRB)
 Tumor suppressor protein
 Restricts DNA replication by preventing cell cycle
progression from G1 phase into S phase.
pRB is active when not phosphorylated.
 pRB when active binds to E2F
transcriptase which pushes the cell cycle
into the S phase when free.
 pRB/E2F keeps the cell cycle
stalled at G1 phase

29. Retinoblastoma Protein (pRB)

30. Telomeres
 A region of repetitive
nucleotide sequences at
each end of a
chromosome.
 Protect the end of
chromosomes from
deterioration or from fusion
with neighbouring
chromosomes.

31. Telomeres
 As cells replicate the
chromosomes start to lose parts
of their telomeres.
 Ultimately as the telomeres are
chipped off the cells can no
longer replicate and they enter
into senescence (grow old).

32. Telomeres

33. Telomerases
 Called also terminal transferase and is
made up of proteins and RNA.
 replenishes telomere nucleotide
sequences at the end of existing
chromosomes.
 This replenishment is achieved
by a process called reverse
transcriptase.

34. E6 & E7 Oncoproteins
 The oncogenic activities of E6 and E7 have
been documented extensively.
Expression of high risk E6 and E7 in primary
human keratinocytes facilitates their
immortalization.
High risk HPV E6 and E7 immortalized
cells display morphologic features of
high-grade intraepithelial lesions
which are well established precursors
of cancers.

35. E6 Oncogenic Activities
 HPV depends on host cell DNA
replication for its survival and
propagation.
 Overexpression of p53 inhibits
viral replication.
 Therefore, high-risk E6 most
important function is to bind
p53 and deactivate it.

36. E6 Oncogenic Activities
 High risk E6 proteins activate telomerase
(a complex enzyme that contains its own
RNA) which is responsible for replicating
telomere DNA at chromosomal ends.
 E6 up regulates telomerase activity
by activation of the human
telomerase reverse transcriptase
that creates single-stranded DNA
using single-stranded RNA at the end
of chromosomes.

37. E7 Oncogenic Activity
 E7 associstes with retinoblastoma
gene product pRb to facilitate
progression of the cell cycle.
 Normally, pRb is bound with E2F
transcription factor forming a
complex that acts as
transcription suppressor

38. Summary of Oncogenic Activities of
E6 and E7
 E6:
 Cell immortalization
 P53 degradation.
 Anti-apototic Effect.
 Genomic instability.
E7:
 Cell immortalization
 Interaction with pRb.
 Telomerase activation.
 Transactivation of E2F.
 Genomic instability.

39. High Risk vs. Low Risk HPVs
Why only high risk HPV viral
types cause cancer while the
low risk HPVs develop
benign lesions?

40. Molecular Testing
for of HPV DNA

41. Human Papilloma Virus DNA testing
 The identification of HPV as the causative
agent of cervical cancer and its precursor
lesions lead to the development of
molecular methods for the diagnosis of and
screening for HPV infections.
 The most common diagnostic
molecular techniques for HPV
detection are:
 Target Amplification
(PCR): Cobas®.
 Signal Amplification
(HC2): Digene™.
 Probe
Amplification:
Cervista®.

42. Molecular Assays for HPV Detection
 Most HPV assays target the L1
region for amplification and
detection.
 L1 gene codes for viral capsid
protein.

43. Target Amplication Assay: Polymerase
Chain Reaction (PCR)
 The most well-known example is the polymerase chain
reaction (PCR).
 PCR can take a single double-stranded DNA and
amplify it to 1 billion copies after just 30 cycles.
 PCR is an easy, cheap and reliable method to
repeatedly replicate a focused segment of DNA.
 PCR relies on thermo cycling that involves
repeated cycles of heating and cooling to drive
reaction forward.

44. PCR (cont’d)
 Components of PCR
reaction:
 Target DNA.
 Themostable DNA
polymerase (Taq Polymerase)
 Two primers (complementary
to the 3’ ends of each DNA
strand)
 Buffer solution, bivalent
cations (Mg++, Mn++),
monovalent cations (K+)
 Nucleotides
 Assay Steps:
1. Denaturation
(94-98° C)
2. Annealing
(50-65°C)
3. Extension
(75-80°C)

45. PCR (cont’d)

46. Signal Amplification Assay:
Hybridization Capture 2 (HC2)
 Specimen hybridized with
specific HPV RNA.
 RNA:DNA hybrids are
captured onto the surface of
a microplate well with
antibody specific to hybrids.
 Hybrids react with ALP
conjugated antibody.
 Chemiluminescent ALP
substrate is added to
reaction and is cleaved by
the enzyme.
 Light emitted is measure by
a luminometer as relative
light units (RLU).
 RLU are measured against
a cut-off value (CV):
 RLU> CV: DNA present.
 RLU< CV: DNA absent.

47. Probe Amplification Assay: Ligation
Chain Reaction (LCR)
 LCR is a method of DNA amplification that
amplifies the probe rather than the target DNA.
 Two pairs of probes are used one for each
strand and are then ligated to form a single
probe.
 LCR uses both a DNA polymerase and a
DNA ligase enzymes to drive the reaction.
 The probes are designed to match two
adjacent sequences of a specific target DNA.
 The probes are attached to radioactive
substances or tagged with a dye for easy
detection of target sequence.

48. LCR (cont’d)
1. Denaturation
2. Annealing
3. Polymerization and
Ligation

49. THE END
“EDUCATION IS THE
KINDLING OF A FLAME
NOT THE FILLING OF A
VESSEL”

50.  References:
1. Arney A, Bennett K M. Molecular Diagnostics of Human Papillomavirus. Lab Med. 2
010; 41(9): 523-530. [www.medscape.com/viewerarticle/727399_print].
2. Bosch F X, Lorincz A, Munoz N, Meijer C J L, Shah K V. The Causal
Relation Between Human Papillomavirus and Cervical Cancer. J Clin
Pathol 2002; (55): 244-265. [www.jclinpath.com].
3. Boulet G, Horvath C, Vanden Broeck D, Sahebali S, Bogers J. Human
Papillomavirus: E6 and E7 Oncogenes. Int J Biochem Cell B 2007 (39):
2006-2011. [www.sciencedirect.com].
4. 4. Farah H, Xu S, Rao S, Winston R. A Brief History of Cervical Cancer.
https://www.globalwomenshealthtechnologies.com/. Retrieved on 23
Apr 2017.
5. 5. Faridi R, Zahra A, Khan K, Idrees M. Oncogenic Potential of Human
Papilloma Virus and its Relation With Cervical Cancer. Virol J 2011 (8):
269. www.virologyj.com/content/8/1/269.
6. 6. Munger K, Baldwin A, Edwards K M, Hayakawa H, Nguyen C L, Owens
M, Grace M. Mechanisms of Human Papillomavirus-Induced
Oncogenesis. J Virol. 2004: 11451-11460. [http://jvvi.asm.org]
Downloaded on March 28, 2017.
7. 7. Wiedmann M, Wilson W J, Czajka J, Luo J, Barany F, Batt C A. Ligase
Chain Reaction (LCR)-Overiew and Applications. [Downloaded from
http://genome.cshlp.org/content/3/4/S51.full.pdf+html].

51.  Video Material:
1. Blackburn, E. [iBiology].(2010, Mar,23). The Roles of Telomeres and Telomerase (Part I) [video file].
Retrieved from https://www.youtube.com/watch?v=5PU_jZwt8KY.
2. Blackburn, E. [iBiology]. (2010,Mar, 23). Telomeres and Telomerase in Human Stem Cells and Cancer (Part
II) [video file]. Retrieved from https://www.youtube.com/watch?v=zqMoDdHWFBA.
3. dmflyboy. (2011, Oct, 22). The Cell Cycle and Cancer: Tumor Suppressor Genes.[ video file]. Retrieved from
https://www.youtube.com/watch?v=MWfv7vfzSEE.
4. Lynch, E. [Elizabeth Lynch]. (2013, Nov ,2). Using Odds Ratios in Case Control Studies. [video file].
Retrieved from https://www.youtube.com/watch?v=wJXaroDs9oo.
5. MacMaster Evidence-Based Practice Workshops. (2014, Apr, 15). Understanding odds Ratios. [video file].
Retrieved from https://www.youtube.com/watch?v=fkWJWKu0FUc.
6. Shaneyfelt, T [Terry Shaneyfelt]. (2016, Feb, 20). How to Interpret and Use a Relative Risk and Odds Ratio.
[video file]. Retrieved from https://www.youtube.com/watch?v=FZzm3-RRlI4.
7. Shomu’s Biology. (2015, Oct, 13). Ligase Chain Reaction (LCR). [video file]. Retrieved from
https://www.youtube.com/watch?v=L3H6A-wN42E.