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BIO DATA
• Nama : Dr. dr. Sri Hartini SpPK (K) , MARS• Alamat : Inst Pat Klin RS Kanker Dharmais
Jl. Let Jen. S. Parman Kav 84-86, Slipi Jakarta Barat e-mail : [email protected]
• Riwayat Pendidikan :– Dokter Umum , FKUGM , 1973– Spesialis Pat.Klin : FKUI, 1985– Magister Administrasi RS : Fak. Pasca Sarjana UI, 1999– Doktor FK UGM, 2015
• Riwayat Pekerjaan :– Peneliti Keselamatan Radiasi & Kedokteran Nuklir BATAN 1985-1993– SMF Pat. Klin RS Kanker Dharmais (RSKD) 1993- sekarang– Ka. Instalasi Patologi KLinik RSKD 2001-2002– Direktur Penunjang Medik RSKD 2002 – 2006– Direktur Umum & Operasional RSKD 2006-2008– Dosen Program Biomedik Kekhususan Onkologi FKUI di RSKD
2008 – sekarang• Qrganisasi :
– PDS. PATKLIN ; Perhimpunan Onkologi Indonesia
MOLECULAR HPV :EARLY DETECTION IN CERVICAL
CANCER
Sri HartiniDharmais Cancer Hospital
HPV classification based on the nucleotide sequence of the capsid protein L1 gene.
Burd Clin. Microbiol. Rev. 2016;29:291-319
Risk classification HPV types
High-risk 16, 18, 31, 33, 35,
39, 45, 51, 52, 56
58, 59, 68, 73, 82
Probable high-risk 26, 53, 66
Low risk 6, 11, 40, 42, 43, 44
54, 61, 70, 72, 81, CP6108
Undetermined risk 34, 57, 83
Alpha Papillomaviruses
Journal of Clinical Virology 32s (2005)
Global Prevalence of HPV16 and HPV18
1. de Sanjose S, et al. Lancet Oncol 2010; 11:1048-1056. 2. Munoz N, et al. Int J Cancer 2004; 111:278-285.
PERSISTEN HR-HPV INFECTION
• Head n Neck (cavum oral, tonsil, oro-pharynx,larynx )
• Skin • Breast
• Cervical
Risk Factor for malignancy
Ten most frequent HPV oncogenic types among women with invasive Cervical cancer by histology in Indonesia
ICO HPV Information CentreInstitute Catala d´Oncologia
Version posted on www. hpvcentre. net in February26th,2016
*No data available. ; No more types than shown were tested or were positive.
ICO HPV Information CentreInstitute Catala d´Oncologia
Ten most frequent HPV oncogenic types among women with invasive Cervical cancer by histology in Indonesia
Version posted on www.hpvcentre.net in February 26th,2016
0
10
20
30
40
50
60
70
80
90
100
Normal Ascus NIS 1 NIS 2 NIS 3 Ca Cervix
%
38.6%
18.6%
17.1%
10.0%
15.7%
HPV 16
HPV 18
HPV 52
HPV 45
Others
% HPV type in Cervical Ca
% HR-HPV + in biopsy/ Cervical swab
Data Dharmais Cancer Hospital 2009-2010
0
2
4
6
8
10
12
14
16 HPV 16+
HPV 18+
HPV 28+
HPV 45+
HPV 51+
HPV 52+
HPV 82+
HPV 16+ dan 18+
HPV 16+ dan 28+
HPV 16+ dan 51+
HPV 16+ dan 52+
HPV 16+, 18+, dan 51+
HPV 16+, 18+, 45+, dan 52+
HPV 18+ dan 31+
Data Dharmais Cancer Hospital 2014
6
5
8
0
6
0
4
3
1
0
1
2
3
4
5
6
7
8
9
HPV 16+ HPV 18+ HPV Multi tipe
Squamous Ca
Adenokarsinoma
NIS 2/3
Data Dharmais Cancer Hospital 2014
P = 0.014
British Journal of Cancer 99, 214-18, 2008
First lesion
Immune response
Incubation1–6 months
Active growth(3–6 months)
Hostcontainment(3–6 months)
Sustained clinical remission 8-30 M
9,8 months
Infection Seroconversionaverage time 9 months
DNA-ve
DNA-ve
DNA+ve
75-90%
Persistent or recurrent disease
Natural history of HR-HPV infection to Cervical Ca
High grade lesion
Invasive Cancer
Modified from Stanley M. Vaccine 2006;24S1:S1/16–22.
(2). Molden T, et al. Int J Cancer.2005:973-6. (3) Rozendaal L, etal. Int J Cancer 1996;68:766-9 (
4).Franco EL, etal. J Infect Dis 1999;180:1415-23. (5)Munoz N, etal. J Infect Dis 2004;190:234-42
4 yrs
2 yrs
7-8 years classically 15 years
Re-infection10-25%
2,38,9-14,8 months 4,5
PERSISTENT ONCOGENIC HPV
HPV 16,1831,45,52
Clinical Science www.clinsci.org Clin. Sci. (2006) 110, 525-541
A. Uninfected Epithelium
Feedback
Transactivation E2F
Basal LayerUpregulation
of Genes
necessary
for S-phase
progressionRegulated
MDM
Regulation
of
levels
P
Cyclin/cdk
P16
MCMPCNAKi67
P14 ARFCyclin E
P pRb
pRb
E2F
pRb
p53
pRb
E7
P16
MCMPCNAKi67
P14 ARFCyclin E
Upregulation
of Genes
necessary
for S-phase
progression
Transactivation
Basal +
Parabasal Layer
E2F
degradationInactivation
of MDMp53
Upregulation
of p53
p53
High p21
Low E7
Low p21
High E7
P21, E7 and cyclin E form a
complex. Cyclin E/cdk inactive
and present at high levels
E7 p21cdk
Cyclin
E7 p21
cdk
Cyclin
p21 + E7 form a complex
p21 inactivated.
Cyclin E/cdk active and present at low levels
CELL CYCLE
PROGRESSION STALLED
S-PHASE
PROGRESSION
B. High Risk HPV INfection
No
Feedback
Feller L, Wood NH, Khammissa RA, Lemmer J - Head Face Med (2010)
Characteristics of HPV test technologies
Characteristics of HPV test technologies
Characteristics of HPV test technologies
NON PCR HPV DETECTION : SIGNAL AMPLIFICATION
1. Release and denature DNA 2. Hybridize RNA probe
with target DNA3. Capture RNA:DNA hybrids
onto a solid phase
4. React captured
hybrids with multiple
Ab conjugates5. Detect amplified chemiluminescentsignal
• Qualitative in vitro test, detect amplified chemiluminecent
• Amplification of signal DNA by HC technology
• Detects 13 high risk anogenital HPV genotypes:16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68
• First in the market• Very manual , Hands on time, Time
to result, No internal controls
• Qualitatif invitro test• Discrimination of 37 genotypes 37 anogenital types:
(6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, and CP6180)
• Discrimination of “high risk” versus “low risk” genotypes
• Suited for detection of multiple infections & epidemiology studies
• Low and high beta-globin reference lines assess cellular adequacy, extraction and amplification for each individually processed specimen
PCR HPV DETECTION : TARGET AMPLIFICATION
LINEAR ARRAY HPV Genotyping Test Result Interpretation
Numerical orderof genotypes
Reference Line
Low Globin ControlHigh Globin High
High Globin Control
Reference Line
GT 18
GT 18GT 31
GT 45
HPV Strip Reference Guide
Low globin control
GT 16
High globin control
Hasil HPV Genotyping : HPV 16, 18, 42, 52/33/35/58, 58
31
35
45
52
58
66
33
39
51
56
59
68
HR 16 18 Contr.
Ch1 Ch2 Ch3 Ch4
Clinical Design
Pooled result for 12 HPV genotypes
• Responsible for ~30% of cervical cancers
Individual result for HPV16
• Most aggresive genotype
Individual result for HPV18
• HPV18+ disease often missed by cytology
• Most common in ADC, the most aggressive form of cervical cancer
Multi colour, single tube Test
Real Time PCR : three HPV results in a single test
• Identification of oncogenic HPV types
• Primary cervical cancer screening
• Different relative risk of cervical cancer according to HPV types
• Essential to investigate the efficiency of the vaccines
• Confirm the unclear cervical cytology result
• Decrease the use of colposcopy
CLINICAL APPLICATION
J Gynecol Oncol. 2016 Mar;27(2):e21
Choi YJ, Park JS Clinical significance of HPV genotyping
Main characteristics of cervical cancer screening in Indonesia
HPVDNA testing is being introduced as an adjunct to cytology screening(co testing) or as the primary screening test to be followed by a secondary, more specific test, such as cytology (in private sector).
Principles for screening programs
Wilson, Jungner WHO Chronicle Geneva. 22(11):473. Public Health Papers, #34. 1968
Principles for screening programs
Principle –Test is accurate and reliable
ATHENA Results: Variability of Cervical Cytology
*To detect ≥CIN2Wright, et al. IntJ Cancer 2014 134(8):1835-43
• ATHENA : Addressing THE Need for Advanced HPV Diagnostics
• A prospective study of >47,000 women
• Atypical Squamous Cells of Undetermined Significance.
Principle –Test is sensitive
Principles for screening programs
Sensitivity of Cervical Cytology (for ≥CIN2)
Whitlock et al. Ann Intern Med. 2011; 155:687−697, W214−5.
Principle –Test is specific
Principles for screening programs
Specificity of Cervical Cytology (for ≥CIN2)
Whitlock et al. Ann Intern Med. 2011; 155:687−697, W214−5.
Up to 33%Cervical cancer cases
are found in women withNormal pap smear
Limitations of cytology
1. Castle PE, et al. Lancet Oncol2011; 12:880–890 plus supplementary tables. 2. Wright TC et al. Int. J. Cancer 2014; 134:1835–1843. 3. Herzog TJ & Monk BJ. Am J ObstetGynecol2007; 197:566–571.
Cervical cancer screening is not the same as HIV screening
HPV Test Sensitivity in Cervical cancer screening
Kinney, et. al., Am J Clin Pathol2010;134:193-199
Requirements of HPV tests
1. The candidate test should have a clinical sensitivity for ≥CIN2 not less than 90% in women of at least 30 years
2. A clinical specificity for ≥CIN2 of the candidate test not less than 98% in women of at least 30 years of age.
3. Should display intra-laboratory reproducibility and inter-laboratory agreement with a lower confidencebound not less than 87% Meijer, et. al., Int. J. Cancer: 124, 516-20 (2009)
ACS/ASCCP/ASCP Guidelines: “the sensitivity of HPV testing for CIN3+ and CIN2+ should be greater than or equal to 90%...”
Saslow et. al., CA CANCER J CLIN 2012;62:147-172
Risk of CIN3+ After Negative Screening Test & European follow-up studies; 24,295 women
Months of Follow-up
Dillner et al. BMJ 2009;377
Risk of CIN3+ After Negative Screening Test ATHENA study : 42.209 women > 25 yrs
cobas HPV Test – Package Insert Months of Follow-up
Co-testing HPV Screening Algorythme
NILM : Negative for Intraepithelial Lesion or Malignancy
•Cotesting is inefficient –It requires two screening tests every time a women
screened
•Cotesting isn’t logical –It combines a relatively insensitive test with a highly
sensitive test
•Cotesting is complicated -It incorporates cytology as part of the initial screen and
cytology-based screening has become incredibly complicated over the last decade
Limitations of Cotesting
HPV primary screening trials
Results from European trials
Ronco et al. Lancet pub online, 2013.
Primary HPV Screening Algorythme
SGO & ACCP Interim Guidance
Huh W et al. Gyn. Oncol. 2015 NILM : Negative for Intraepithelial Lesion or Malignancy
HPV Primary Screening SGO & ASCCP Interim Guidance
•Because of equivalent or superior effectiveness, primary hrHPVscreening can be considered an alternative to current US cytology-based cervical cancer screening methods. Cytology alone and cotesting remain the screening options specifically recommended in major guidelines
•Based on limited data, triage of hrHPV-positive women using a combination of genotyping for HPV 16 &18 and reflex cytology for women positive for the 12 other hrHPV genotypes appears to be a reasonable approach to managing hrHPV-positive women
Huh W et al. Gyn. Oncol. 2015
Society of Gynecologic Oncology, American Society for Colposcopy and Cervical Pathology, American College of Obstetricians and Gynecologists, American Cancer Society, American Society of Cytopathology, College of American Pathologists, and the American Society for
Clinical Pathology
•Re-screening after a negative primary hrHPV screen should occur no sooner than every 3 yrs.
•Primary hrHPV screening should not be initiated before 25 years of age.
•They note that primary hrHPV screening at age 25-29 yrs may lead to increased CIN3 detection but impact of increased number of colposcopies, etc needs further investigation.
HPV Primary Screening SGO & ASCCP Interim Guidance
Huh W et al. Gyn. Oncol. 2015
• an option to reduce costs and increase patient participation in HPV screening programs.
• Results using self-and clinician-collected samples showed equivalent HPV genotype distributions and prevalence
• Feasible and well accepted, and showed sensitivity and specificity comparable to those achieved using clinician-collected samples .
• Self-testing detected precancerous cervical lesions even earlier than cytology . Home-based HPV testing is a good alternative not only for people residing in developed countries, but also for people living in developing
HPV GENOTYPING : SELF COLLECTING SAMPLES
Choi YJ, Park JS J Gynecol Oncol. 2016 Mar;27(2):e21
J Gynecol Oncol. 2016 Mar;27(2):e21Choi YJ, Park JS
Therapeutic vaccine using HPV 16-specifc CD8+ T-lymphocyte responses that stimulates the expansion of CD8α+lymphoid dentritic cells and facilitated the expression of HPV antigen through the MHC I pathway.
TAKE HOME MESSAGES
• Cervical cancer is caused by 14 "high-risk" types of HPV • Indonesia : Common type hr-HPV : 16, 18, 45, 51/52• hr HPV detection :
• RT-PCR with internal control more sensitive than non PCR method
• Utilization of hrHPV testing for screening :• identifies BOTH women with disease today and • women at risk for developing disease in the future• HPV 16/18 stratification is important & has high medical value
• Screening Cervical Cancer : • Cytology alone is not sufficient
• Co testing HPV- DNA & Trial Primary HPV-DNA• Future application : Self sampling & Therapeutic vaccine