head and neck squamous cell carcinoma and hpv
TRANSCRIPT
Head and neck squamous cell
carcinoma and HPV
Pr Cécile Badoual
Pathology dpt,
Hôpital Européen G
Pompidou, Paris
Incidence Head & Neck Cancers
World : 500 000 / year
US: 38 500 / year
France: 16 000 / year
Evolution of Incidence
Men : 13 000 (-), women : 3000 (+)
Alcohol and tobacco
Bad prognostic survival after 5 years 50%
Surgery, chemo and radiotherapy
Epidemiology
Head and neck cancer classification (OMS 2005)
Squamous cell carcinoma Verrucous carcinoma Basaloid carcinoma Papillary squamous cell carcinoma Spindle cell carcinoma Acantholytic squamous cell carcinoma Adenosquamous carcinoma Lymphoepithelial carcinoma (UCNT)
• Oral cavity • Nasopharynx • Oropharynx • Larynx
Giant cell carcinoma Salivary-type tumor mucoepidermoid carcinoma
Adenoid cystic carcinoma neuroendocrine tumor typical carcinoid atypical carcinoid Small cell carcinoma Mixed small cell carcinoma
•Oral cavity • Nasopharynx
• Oropharynx • Larynx
Head and neck cancer classification (OMS 2005)
Squamous cell carcinoma of the head and neck Prevalence: 90% of tumors Nasal and sinus: 80-85% Nasopharynx: 75% Oropharynx and hypopharynx: 89% Larynx: 99% Oral cavity: 90%
Head and neck squamous cell carcinoma
Normal epithelioma
Tumor
Inflammatory
infiltration
Head and neck squamous cell carcinoma (HES)
Laryngeal SCC : Evolution of the incidence in
France
Evolution des cancers du larynx
chez l'homme entre 1975 et 1995
0
5
10
15
20
25
75 77 79 81 83 85 87 89 91 93 95
Années
Tau
x s
tan
dard
isé p
ou
r 100 0
00 h
ab
itan
ts
Incidence Mortalité
Evolution des cancers du larynx
chez la femme entre 1975 et 1995
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
75 77 79 81 83 85 87 89 91 93 95
Années
Tau
x s
tan
dard
isé p
ou
r 100 0
00 h
ab
itan
ts
Incidence Mortalité
Hyperplasia Dysplasia Dysplasie
in situ
Carcinoma
Dysplasia Dysplasia
Carcinoma Carcinoma
Normal mucosa Hyperplasia Dysplasia Carcinoma
9p21 LOH
inactivation p16
3p21, 17p13 LOH
mutation p53
11q13, 13p21, 14p32 LOH
amplification cycline D1
6p,8,4q27, 10q23
LOH
inactivation pTEN
in situ carcinoma
Forastiere NEJM Forastiére NEJM
Environmental (polycyclic hydrocarbons ...)
Marijuana, opium, betel (India)
Gastroesophageal reflux
Genetic and family background
Chronic inflammation in mouth (lichen lesions ..)
Role of new materials used in dental amalgam: discussed
Protective role of fresh fruits and vegetables (vit C and antioxidants)
HPV
Risk factor except Alcohol and tobacco
HPV and cancer
Very high incidence of HPV infection over the world (660 million people infected worldwide) Increased prevalence in developing countries and low socioeconomic status E6 and E7 inhibit the function of tumor suppressor genes p53 and retinoblastoma protein 5% of cancer are due to HPV : cancer of the cervix (++), anal cancer (an increase in immunocompromised patients), cancer of the vulva, vagina and penil.
Pathogenic role in head and neck cancers suspected since the early 1980s (Syrjänen K et al. Int J Oral Surg 1983) Meta-analysis of 5046 patients with head and neck cancers found the presence of HPV in all localizations 25.9% of cases (Kreimer AR et al Cancer Epidemiol Biomarkers Prev 2005) confirmed by other studies (Mork et al N Engl J Med H 2001) In non-smoking, HPV found in 40% of these cancers (Fouret et al Arch Otolaryngol 1997) Among the head and neck cancers: Oropharynx + + (35%) Tonsil: 50-65%
HPV and cancer : epidemiology
• "Classic"
Males: 55 - 65 years
Smoking and alcohol intoxication (15
x the risk of head and neck cancer)
low socio-professional level
p16 gene mutated
• "Emergent" increasing
younger: 40 years
Incidence female subjects larger
not related to alcohol and tobacco
intoxication
Oropharynx
Clinical entities in HNC
Marur, Lancet Oncol 2010
HPV + carcinoma : an increasing entity
Retrospective E6 and E7 mRNA PCR in
biopsies of cancer in the Stockholm region
Increased frequency of cancers of the tonsil
factor 2.8
In more than 80% the serotype HPV16 is
found
date % HPV in tonsil cancer
1970 23
1980 29
1990 57
2002 68
Hammarstedt ,
Acta Otolaryngol 2007
Tonsil cancer
Cohort study of 12 French hospitals
Retrospective: 2000-2009 314 biopsies HPV in 57% oropharynx 46% in the oral cavity HEGP to 71% and 0% Age: no difference
female> male HPV 16: 89%
Lacau St Guily J, Head Neck Oncology 2011
Oropharyngeal cancers in France
L1: envelope
protein that
allows the
internalization
of the virus.
Targeted
prophylactic
vaccines
against HPV
E6 and E7 are involved
in carcinogenesis.
Essential for the
immortalization of the
cell
Target therapeutic
vaccines as the virus
has infected person
Role of the HPV
DNA virus with more than 120 subtypes, some high-risk oncogenic:
16 + +, 18 +, 45, 31, 33, 52, 58, 35
Internalisation of the HPV
Traffic cytoskeleton to reach the nucleus Capsids larger than the nuclear pores (29nm) Required disassembly of the capsid Once the replicated viral genome is in the nucleus, a newly synthesized viral proteins come from the cytoplasm to the nucleus where virions are formed
HPV non-lytic virus
Release of virions in death cell and during desquamation of the squamous cells E4 protein by degrading the cytokeratins, facilitates the release of virus
HPV infection history
The virus infects keratinocytes in the basal layer of the epidermis. The virus
particles assemble at the surface layers of the epidermis. Little or no
cytopathic effect, and cytolysis in the HNSCC
HPV replication
Tissue specificity in vitro and in vivo unclear Non-dependent cell receptor (ubiquitous) transcriptional specificity
Keratinocyte differentiation essential to the viral cycle Limiting factor in vitro models and Culture
Distribution over the world
Distribution by continent
HNSCC and HPV : clinic
- Patient non-alcohol and tobacco
More balanced ratio of male / female
Basaloid tumor type
- Non-mutated P53
- Cancer often with low tumor volume (T1-T2) and nodal
basaloid squamous cell carcinoma conventional squamous cell
carcinoma
– Sexual behaviors – Head and Neck Cancers
D’Souza G, NEJM 2007
Gillison ML, JNCI 2008
– HPV Prevalence • Meta-analysis, 5681 patients
• Prevalence of HPV: 22%
• Prevalence of HPV in Oropharynx cancers: 41%-100% (Sweden..)
• HPV 16: 87%
• HPV 18, 33.
Dayyani F, Head Neck Oncology 2010
HPV – Oropharynx carcinomas
Standardization of sampling
Techniques
validation
Cuschieri & Wentzensen, 2008
HPV detection :limits
HPV diagnostic
Serology: Many false negatives
HPV is a virus that does not cause cytolysis is no danger signal for the
immune system. No viremia.
Pro
po
rtio
n o
f w
om
en
wit
h
sero
co
nvers
ion
HPV16
HPV18
HPV DNA Detection isolated
Carter et al J Inf Dis 2000
1 month after first detection HPV
In women with cancer of the cervix,
40% were seropositive anti-L1
HPV DNA Detection repated
Screening test for the detection of HPV DNA
Principle: Use cocktail of primers to amplify multiple HPV
genotypes
Ex Abbott Real Time High Risk HPV (real-time PCR) Hybrid
Capture 2 (Qiagen / Digene)
Genotyping of HPV testing
Principle: PCR and reverse hybridization on strips
Ex INNO-LiPA HPV Genotyping Extra: Detection of HPV
serotype 28 serotypes (Innogenetics)
Linear Array HPV Genotyping Test: Detection of HPV
serotypes 37 (Roche)
paraffin and frozen section
Molecular techniques:
PCR (Technical Reference)
Molecular techniques: in situ hybridization (DNA)
In situ hybridization (ISH), is based on the use of a mixture of probes (DNA)
directed against oncogenic HPV (16,18,32,45,...)
Interest: to demonstrate the integration of the virus in tumor cells and prove
the causal link between cancer and HPV infection (paraffin and frozen)
Good agreement between PCR and ISH ,even if better sensitivity of PCR
is proved (Shi J Clin Oncol, 2009)
HPVnégatif HPV positif
Molecular techniques: in situ hybridization ( E6/E7 mRNA)
HES
HIS ADN
HIS E6/E7 mRNA
IHC p16
Immunochemistry
Antibodies against HPV are not specific, used in
some articles. Not used routinely
P16 antibodies:
Opportunity to see an accumulation of the protein
during overexpression in the cell.
P16 (CDKN2A) and cancer
P16 and cell cycle regulating G1-S
P16 and carcinogenesis
P16-RB control
locus P16, CDKN2A
A gene (9p21), two transcripts: p16 and p14ARF
p16 is a regulator of the way Rb
p14ARF is a regulator of the p53 pathway
Chr 9p
ARF
MDM2
TP53
P16
Cycline D/CDK4-6
RB
cellular cycle
control
Cycle
G1
S G2
M
G0
CDK4/6
CyclineD
CyclineD
CDK4/6
Checkpoint G1-S P16
Cycle cellulaire and transition G1/S
RB
E2F
RB
E2F
P16 is a negative regulator of cell cycle
The regulatory mechanism of the transition G1-S is almost
always altered in the cancer cell
Activators hyperactive or expressed
Inhibitors inactivated by deletion, mutation, degradation or
interaction with cellular proteins or viral (HPV….)
Cancer et transition G1/S
P16 (CDKN2A) suppressor gene tumor
Activation of oncogenes (ie CDK, cyclin ...)
tumor suppressor
genes inactivation
•Activation of
proliferation
amplification
translocation
activating mutations
•Inhibition of proliferation
biallelic alterations
inactivating mutations
deletions
epigenetic
P16 is a suppressor gene tumor
Yuan J. Pathol.189:358-362 (1999)
Rb-
P16+
Rb+
P16 is frequently overexpressed in Rb- tumor
P16
Absent
CDK4/6
Cyclin D
Cyclin D
CDK4/6
Checkpoint G1-S
P16
Cellular cycle : G1 / S transition and HPV +
cancer
RB
E2F
RB
E2F
+
E7
HPV infection
Rb Inactivation
p16 expression in HN mucosa
p16- normal tonsil
p16 HN dyplasia
Study of p16 in a series of 60 cases of dysplasia or healthy
mucosa (Am J Surg Pathol Gologan, 2005)
Expression of p16 in head and neck
normal mucosa
• There is a marked, discontinuous, heterogeneous and
mainly basal staining
• Study of p16 in a cohort of 262 tonsils (normal or
hyperplasia), 28% were p16 + in the crypts and germinal
centers. No staining in non-cryptic epithelium.
1% of the sample have a PCR HPV + ... ...
Other mechanisms involved in cell cycle? (Klingenberg,
Histopathology 2010)
P16 expression in HNC
Interprétation des résultats
p16- squamous cell carcinoma
HIS HPV- squamous cell carcinoma
Expression of p16 according
to histological types of HNC
p16- basaloid squamous cell carcinoma
Basaloid squamous cell carcinoma and HPV
Study of p16 and HPV expression in 28 basaloid squamous
cell carcinoma (Chernock Human Path 2010)
Basaloid squamous cell carcinoma, particularly
oropharyngeal is a separate entity in which HPV is closely
associated with prognosis (Thariat *, Badoual* J Clin Pathol
2010)
Expression of p16 in lesions
associated with a low-risk
oncogenic HPV
Expression of p16 in lesions due to
HPV11 and HPV6
HPV 6 and 11 are weakly oncogenic, but are found in
oropharyngeal cancers. p16 staining can be positive for
these patients (Syrjänen, Ann Oncol 2010)
HPV: good prognosis factor in oropharyngeal
squamous cell carcinoma
oropharyngeal cancer are associated with HPV
. a better prognosis
(Licitra LJ Clin Oncol 2006, Fakhry CJ Natl Cancer Inst
2008, Gillison ML J Clin Oncol 2009, Rischin FJ Clin Oncol
2009, Kian Ang NEJM 2010)
. more radiosensitive and chemosensitive
(FP Worden J Clin Oncol 2008, Fakhry CJ Natl Cancer Inst
2008)
... Than the oropharyngeal cancers unrelated to HPV
P16: good prognostic factor of these cancers?
Ang NEJM 2010
Correlation between the expression of P16
and the detection of HPV by ISH and PCR
Lewis JS,
Am J Surg
Pathol 2010
From 10 to 20% of p16 + carcinomas are HIS and PCR-
Role of p16 in the cell cycle?
Pronostic :
P16+/HIS+
and
P16+/HIS-
>>>>>
P16-/HIS-
Prophylactic vaccination against HPV could
eventually reduce the incidence of these cancers
L1-VLP vaccine (virus-like particles:
assembly of capsid proteins of the
virus)
Gardasil (HPV 16 VLP-L1, 18, 6 and
11)
Vaccine Cervarix (HPV16 L1 VLP-and
18)
Technical Committee on Immunization has recommended this vaccination
for girls at the age of 14.
Because of the head and neck cancers associated with HPV, it would also
be desirable to extend the vaccination to boys.
Therapeutic HPV vaccine becoming?
Rational development of therapeutic HPV vaccine:
The prophylactic vaccine is expensive and coverage is low
600 million people are already infected against HPV in the world and
prophylactic vaccines are not effective in case of pre-existing infection
against HPV. Preventive
vaccines seek to
induce Ab against
L1 capsid protein
to prevent the
entry of the virus
Therapeutic vaccines attempt
to induce LT cytotoxic against
the viral proteins E6 and E7
expressed by transformed
cells.
L1
Ab
T cell
HPV infection associated with a good pronosis
(survival, disease free survival)
HPV+
HPV-
Overa
ll s
urv
ival
(%)
0 10 20 30 40 50 60 70
0
10
20
30
40
50
60
70
80
90
100
Time (months)
P = 0.004
A
P = 0.003
HPV+
HPV -
Lo
co
reg
ion
al c
on
rol (
%)
B
0 10 20 30 40 50 60
Time (months)
10
20
30
40
50
60
70
80
90
0
100
64 patients cohort same TNM and localisation HPV+/HPV-
A Duray, clin dev immunol
2010
APC
CD8
CD4
Tumor
“Danger”
CD40
CD40L
“killing”
B7
CD28
Treg
CD4+
CD25+
Local immuno-suppresion
(non specific)
Cellules tumorales et système immunitaire
CD4 and
FoxP3
64 patients same
localisation andTNM.
Regulatory T cells CD4+Foxp3 infiltrating head
and neck cancer HPV+/HPV-
HPV + HPV -
CD4+FoxP3+ lymphocytes lymphocytes CD4+FoxP3+
immunostaining
The vaccine based on long peptides derived from E6 and E7
proteins of HPV yielded encouraging clinical results in this group of
patients:
Complete regression of lesions VIN III:
in 5 / 20 patients, three months after the last vaccination,
in 9 / 19 patients, 12 months after the last vaccination.
Therapeutic HPV vaccine for patients with
dysplasia grade III vulvar (VIN 3) HPV16
associated (VIN3)
Kenter G N Engl J
Med 2009
3 month after
vaccination Before
vaccination
May Cancer Vaccine efficacy depend on
the site of priming and tumor Location
Route of immunization : Mucosal (intranasal) or
systemic (intramuscular)
Vaccine : Long peptide or STxB-based vaccines
Gal
Gal
Glc ceramide
(Gb3 glycolipid receptor)
Plasma membrane
Cytoplasm
Gb3 is preferentially expressed
on DC
(Haicheur J Immunol 2000, Vingert B Eur J Immunol 2006)
nonreplicative delivery vector
targeting dendritic cells, the B
subunit of shiga toxin
May Cancer Vaccine efficacy depend on
the site of priming and tumor Location
Route of immunization : Mucosal (intranasal) or systemic
(intramuscular)
STxB-based vaccines
Gb3 is preferentially expressed on DC
E7 B
Induction of CTL was observed when STxB was
coupled to E7 (Pere H Blood 2011, Adotevi O J Immunol 2007)
Non
vaccinate
d
mice
STxB-E7
IM
E7
IN
STxB-E7
IN WT
mice
Non vaccinated mice Isotype
STxB-E7
IM IM
STxB-E7 IN
Nu
mb
er
of
CD
8+T
cells/
mm
2
0 20 40
60
80
100
120
IM IN
* *
STxB-E7
Sandoval F, Terme M,
Nizard M*, Badoual C*, et
al Science Transl Med
2013
Intranasal ou intramuscular vaccination with STxB-E7
preserve mice against sub cutaneus TC1 tumour’s spreed
control
STxB-E7 IM
STxB-E7 IN
0 10 20 30 40 50 60 0
10 20 30 40 50 60 70 80
90 100
Time (days)
Perc
en
t su
rviv
al
0 10 20 30 40 0
100
200
300
400
Time (days)
Tu
mo
r s
ize
( m
m2)
Sandoval F, Terme M, Nizard M*, Badoual C*, Science Transl Med 2013
HNSCC and HPV : Conclusions
- A new clinical entity with a clinical presentation and a different
molecular profile of these cancers associated with tobacco and alcohol
More favorable prognosis
- Improved response to chemotherapy and radiotherapy which should
lead to therapeutic trials of reduced doses of chemotherapy and
radiation.
Any evaluation of new therapeutic approaches should be
stratified by HPV status of patients
- Determination of subgroups of patients likely to receive appropriate
treatment for their cancer: radiotherapy, chemotherapy and immunotherapy
with anti-tumor therapeutic vaccination.
Chaturvedi,
JCO, 2011
P16 and HN-perspectives
• Subgroups of patients for appropriate treatment for their
cancer: radiotherapy, chemotherapy and immunotherapy
with anti-tumor therapeutic vaccination.
• Importance of the
microenvironment in head and
neck, the role of regulatory cells
(Badoual Head Neck 2010) particularly
in chronic infections .... As in
HPV + tumors.
• Vaccination for boys? Badoual, Clin
Cancer Res 2006
CD4/Foxp3