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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 : sri.harijanto@gmail.com

• 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