HPV

Molecular

Pa Po Va virus

Molecular

Oncogenesis

Nam Hoon Cho, M.D.

Yonsei Univ Coll Med, Dept of Pathology

Presentation FlowHPV testing guideline: pros and cons

HC-2

Genotyping

HPV genome new highlight-HPV genome new highlight-

E6-E6AP-PDZ binding

E1^E4 and E2^E5

Controversial issue in cytopathology

What is phenotype with ASCUS?

CIN 2 from the viewpoint of HPV

HPV types assessed by the IARC

Monograph Working group

α β1 16 Most potent type 2B 5,8 Limited

1 18,31,33,35,39,45,51,52,56,58,59

Sufficient carcinogen

2A 68 Probably carcinogenic, but limited evidence

2B 26,53,66,67,70,73,82 Possibly carcinogenic,

2B 30,34,69,85,97 Limited evidence

3 6,11

3 Other type

Lancet Oncol 2009;10:321-2

HPV types assessed by the IARC

Monograph Working group

α β1 16 Most potent type 2B 5,8 Limited

HPV 58 PPV for CIN 3+= 54.9% (An HJ et al. Cancer 2003; 97:1672)

HPV frequency: 16/18/58/39,52,56,51 (Cho et al. Am J Obstet Gynecol 2003; 188:56)

1 18,31,33,35,39,45,51,52,56,58,59

Sufficient carcinogen

2A 68 Probably carcinogenic, but limited evidence

2B 26,53,66,67,70,73,82 Possibly carcinogenic,

2B 30,34,69,85,97 Limited evidence

3 6,11

3 Other type

Lancet Oncol 2009;10:321-2

(Cho et al. Am J Obstet Gynecol 2003; 188:56)

HPV with no CIN: 37.2%(Hwang et al. Gynecol Oncol 2003; 90:51)

HPV in Korean commercial sex workers:47% (16, 51) (Choi et al. J Med Virol 2003; 71:440)

HPV multiple infection in 9.1% and OR 31.8 folds for CIN 3+ (Lee et al Cancer Lett 2003;198:187)

Time Line of Cervical HPV

Infections And Progression

to Cervical Cancer

HPV

PrecancerCancer

Rate

15 y.o. 30 y.o. 45 y.o. Age

Adapted from Schiffman & Castle, New Eng J Med 353:2101-4, 2005

Lifetime incidence of genital HPV infection >80% in U.S.

Most infections are asymptomatic and clear spontaneously,

eliminating cancer risk for that infection.

Persistent infection with a high-risk HPV, especially HPV16

or 18, is the single most important risk factor for

progression to precancer and cancer.

Life Cycle of HPV Infection

HPV Infection is usually transient8-14 months

Life Cycle of HPV Infection

Stratified

Virion

Supra-

AssembledVirus

Basement

membrane

Dermis

Stratified

squamous

epithelium Virion

Supra-

basal

cells

Basal

cellsHPV

DNA

HPV DNAreplication

HPV16 Binds

the Basement Membrane

HPV-16 Laminin-5

Mouse vaginal tract: 2 hours after exposure to HPV16

(8 hours after exposure to nonoxynol-9)

Kines, Thompson, Lowy, Schiller and Day, PNAS 106: 20458-63, 2009

Neutralizing

L1 Antibodies

(in red)

Bound to

PapillomavirusPapillomavirus

Particle

VLP Vaccination Induces High

Titer Antibodies that Prevent

Basement Membrane BindingDay et al, Cell Host Microbe 8: 260-70, 2010

Virion

No Infection

Basement

membrane

Dermis

Stratified

squamous

epithelium

Virion

STOP

0 1000 2000 3000 4000 5000 6000 7000 7905 bp

E6

E1E7

E2

E4 L2E5

L1

My 09/11

gp5+/6+

HPV16-1

HPV16-2

Genomic Organization of HPV

• Episomal replication: E1/E2• Host integration:

E1/E2 ORF disruption

E2-DNA dimer: strong > E1-DNA hexamer

E2/E6 real-time PCR

1:episomal >or <1:mixed 0: integration

0 1000 2000 3000 4000 5000 6000 7000 7905 bp

HPV-induced tumorigenesis

1. integration pathway

2. episomal pathway

E1/E2 promote genomic

instability through aberrant

replication of integrated

sequence

L-gene expression >>E-gene

HR vs LR HPVFrom Infection to Tumor InitiationIntegration: 16: >50%, 18: most

vs LR: seldom to never

Transforming activityTransforming activityHR E7: immortalize at a low frequency- CDK2hg/ p21, p27low

vs. LR E7- decreased p21 abrogation

HR E6: no transforming activity, growth arrest abrogation by p53-

E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding, but no p53 degradation

HR E6+E7: highly efficient immortalizing, proliferation and avoid

apoptosis , but not tumorigenicv-ras/v-fos coexpression: tumorigenic

E6: telomerase ex, PDZ domain [ X-S/T-X-V/L/I]-containing protein

(p53-independent target), TNF modulation vs. LR: no PDZ domain

HPV E6- p53 Proteolysis

- No mutation of p53

Both TSG are

impaired by

HPV E6/E7

HPV E7pRB functional inactivation

= hyperphosphorylation

=TF release

=TF activation

=G1-S restriction point entry

E6 and PDZ domain

PDZ-domain protein: scaffolding protein

cell polarity, cell junction

E6 HR/E6AP-PDZ binding induce loss of cell polarity, EMT,

and carcinogenesis.

E2 and CK13 expression

is reverse to P16/CK14/ki67

Xue Y et al. Cancer Res 2010;70:5316-5325

E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)E4

Long-acting multitasking

role of E4 genome

Most divergent domain in sequence according

to HPV types

L1

N terminal: Binding to cytokeratin and concomitant

destabilization of cytokeratin network

Body: cell cycle arrest at G2/M checkpoint

L1 protein encoding

HPV E4functions cytokeratindisruption

L1 Capsid cross-linking in oxidizing

environment of corneum

The more L1 capsid, the less progressive

Virions

E5 Genomic Organization of HPV

E2

L2E56 Cys residue

• Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein

• Weak transforming activity alone, but strong in BPV E5

E2^E5 (HPV 83 aa/ BPV 86 aa)

• Weak transforming activity alone, but strong in BPV E5

– enhancing transforming activity of E6/E7 as oncogenic potential

• EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-

endosomal PH alteration- EGFR turnover alteration

• PDGFR ligand-independent complex during tissue repair after HPV entry

• Interfere with gap junction and alter caveolin-1

• Anchorage-independent growth (anoikis) stimulation (HPV release)

• Inducer of koilocytosis as cofactor with E6, in vitro (Am J Pathol2008; 173:682)

Koilocytosis by E5

Cytoplasmic vaculation-unclear reason,

but contributing to fragility to make it easy

to release viral particlesAm J Pathol 2008; 173:682-8

E5 and E6

induce

koilocyte,

E7 inhibits

koilocyteE7 inhibits koilocytosis

koilocyte

Sequence of events

of HPV genomeE1 E2 E4 E5 E6 E7 L1 L2

Location Cyto-Nc

shuttling

Cyto-

NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-

Nc

Cyto-Nc

Main

action

Replication Replicat

ion

CK-disruption EGFR/PDGFR

Anoikis-

resistance

P53

disruption

pRb mut

P16 ov.ex

Ki67 ov.ex

Major

capsid

Minor

capsid

Sequence #5 #6 (?) #2 #1 #4 #3 #7 #8Sequence

of events

#5 #6 (?) #2

(92a-a)

#1

(16kDa;83a-a)

#4

(18kDa;

150a-a)

#3

(13 kDa;

98 a-a)

#7 #8

Cell cycle G2 arrest S-phase

entry

Oncogeni

c

suggested Key role

(HR vs LR)

Key role

(HR vs LR)

Prime

goal

Replication Control Most abundant

viral product

Lateral spread

Infected basal

cells

proliferation in

viral entry,

Koilocytosis

Immortalize,

but not

alone

Permissive

milieu

Immortalize

Dominant

oncogene

Cross-

linking

VLP-

vaccine

Shape

and

stability

Immortalized E6/E7- no tumorigenic, in vitro E6/E7+V-ras/fos - tumorigenic

Changes in the HPV16 life cycle

during the development of cervical

cancer•• CIN1 generally resemble productive lesionsCIN1 generally resemble productive lesions

•• In CIN2 and CIN3 lesions, the order of life cycle events is unchanged, In CIN2 and CIN3 lesions, the order of life cycle events is unchanged,

but the extent of but the extent of E7 (dominant oncogene) expression is increasedexpression is increased

•• Viral genome integration into cellular DNA. Viral genome integration into cellular DNA.

•• Loss of E2 leads to increased E6/E7 expressionLoss of E2 leads to increased E6/E7 expression

CIN1 CIN2 CIN3

VS

•• Loss of E2 leads to increased E6/E7 expressionLoss of E2 leads to increased E6/E7 expression

• • In cervical ca, the productive stages of the virus life cycle are no longer In cervical ca, the productive stages of the virus life cycle are no longer

supported and viral supported and viral episomesepisomes are usually lost. are usually lost.

Productive vs. Proliferative

CIN2 Where?

Spectrum of Changes in Cervical Squamous Epithelium Caused by HPV Infection.

NEJM, 2003

Replication: E4/E1/E2/L1/L2

Differentiation

LSIL/ CIN I

CIN 2: Heterogeneous spectrum

of Pathology and HPV

Proliferation: E6/E7/E5

(Ki67/ p16/cyclin E/cyclin B1)

Take Home Message

• E1^E4, E2^E5 splicing genome

– Multitasking crucial roles from entry to exit

– Phenotypically meaningful sign of – Phenotypically meaningful sign of

koilocytes and ruffled surface

• LSIL vs. HSIL completely two different

entity- CIN2 should be included in HSIL,

or overlapped with CIN 3

No territory of ASCUS

restricted to PAP for holding Dx

Spectrum compatible with ASCUS

exist in tissue Dx?

Conservativeconcept

Challenging Concept

1 1restricted to PAP for holding Dx

two spectrum of CNI or CIN1

borderline lesion? CIN ½

Replication in low-risk HPV vs.

proliferation in HR-HPV

exist in tissue Dx?

Abortive koilocyte

Queryocyte

Regressing koilocytosis

HR-LSIL further progressive?

LR-HSIL; 0-5%

Multiplicity? Virulence?

CIN2-heterogeneous group

2 2

Revisited ConceptASCUS cells caused by incomplete E4/E5 activity

Abortive koilocyte

Queryocyte

Regressing koilocyte : not enough cleared by immune sys

1

2 HR-LSIL

Need to monitor carefully, but no overconcern!

LR-HSIL

Occur rarely in single, but often combined, need to be further studied

CIN2

definitely heterogeneous in HPV life cycle to manifest combined

productive and proliferative phase

2

3

ASCUS vs. LSIL

What more? How properly? How efficiently?How legally?

Performance characteristicsDesignation Probes/primers

Reaction

product

Analytical

sensitivity, fg

detectable

types

Hybridi

zation

HC2 HPV DNA

assay

Mixture of

RNA probes

DNA/RNA

hybrids25–75 13

PCRMY09/11 Dot blot Degenerate primer 450 bp 0.1–100 39

PCR PGMY09/11 Mixture of 450 bp 0.1 27

PCR PGMY09/11

reverse LBA

Mixture of

consensus primers450 bp 0.1 27

PCR GP5+/GP6+ EIA

ELISA systemConsensus primers 150 bp 0.5–10 20

PCRGP5+/6+ reverse

LBAConsensus primers 150 bp 0.5–10 37

PCRSPF-PCR reverse

LiPA

Mixture of

consensus primers65 bp 0.1–10 43

Iftner T, et al. J Natl Cancer Inst Monogr. 2003;(31):80-8.

The concept of HPV load

with clinical behaviorSnijders P, et al. J Pathol 2003; 201: 1–6.

Analytical VS Clinical SensitivityAnalytical VS Clinical Sensitivity

SnijdersSnijders P, et al. J P, et al. J PatholPathol 2003; 201: 12003; 201: 1––6.6.

비정상비정상자궁경부자궁경부세포검사를세포검사를보인보인환자의환자의처치에처치에있어서있어서 HPV DNA testing HPV DNA testing

�� ASCASC--US/LSIL US/LSIL 환자의환자의처치처치및및추적추적검진검진

–– 고위험군고위험군 HPV DNA testing : HPV DNA testing : 연연 11회회시행시행 급여급여인정인정

�� 조직검사상조직검사상 CIN 1CIN 1으로으로확진된확진된환자의환자의추적추적검진검진

–– 고위험군고위험군 HPV DNA testing : HPV DNA testing : 연연 11회회시행시행 급여급여인정인정

CIN II/III CIN II/III 환자의환자의치료치료전전,,후후검사로서검사로서HPV DNA testing HPV DNA testing

�� 고위험군고위험군 HPV DNA testing: HPV DNA testing:

–– 치료치료전전 11회회시행시행급여급여인정인정

–– 치료치료후후최소최소 66개월개월후후 11회회시행시행급여급여인정인정–– 치료치료후후최소최소 66개월개월후후 11회회시행시행급여급여인정인정

�� 치료치료후후시행한시행한고위험군고위험군 HPV DNA testingHPV DNA testing이이양성양성

–– 66--1212개월개월간격으로간격으로고위험군고위험군 HPV DNA testingHPV DNA testing을을

반복반복시행시행 급여급여인정인정

Take Home MessagesQ1) What is the most abundant genome in LSIL?

1. E6 2. E7 3. E4 4. E5

A1) 3. E4

Q2) What reacts first when infected basal cells?

A2) 4. E5A2) 4. E5

Q3) What is inducing this change?

A3) 4. E5

Q4) What is persistently identified in cytoplasm ?

A4) 3. E4

Q5) What is dominant oncogene?

A5) 2. E7

Despite the common notion that

cervical histology Is the golden standard,

cytology is an important indicator of

subsequent risk of CIN3+ within the

group of normal histology.

Cancer Res 2010; 70:8578-86

Thank youNam Hoon Cho, M.D.

Yonsei Univ Coll Med, Dept of Pathology

Thank you