Multiplex PCR:Multiplex PCR:A Game changer A Game changer

in in infectious disease diagnosticsinfectious disease diagnostics

Multiplex Real Time PCRfor diagnosis of

Human Papilloma Virus Infections

Dr Ashok Rattan,MBBS, MD, MAMS

COO & MDStar Metropolis Clinical Laboratories

Non enveloped double stranded DNA virus: over 100 types15–20 oncogenic30–40 anogenital

L1: major viral capsid protein – immunogenic L2: minor viral capsid protein – immunogenic

Human Papilloma Viruses

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HPV & Cervical Cancer• HPV recognized as the underlying cause of HPV recognized as the underlying cause of

cervical cancer since 1996cervical cancer since 1996

– NIH Consensus Conference on Cervical Cancer, 1996

– World Health Organization/European Research Organization on Genital Infection and Neoplasia, 1996

HPV causes Cx Ca

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Natural History of HPV Infections • Sexually transmitted

• Usually no symptoms• No treatment for HPV infection before symptoms• Immune system clears most cases; some persist

• HPV present in >99% of cervical cancers• High risk types (16, 18) associated with cancer • Low risk types (6, 11) are associated with genital warts • All can cause abnormal Pap tests

Human Papillomavirus. ACOG Practice Bulletin No. 61. 2005; 105: 905-18.

HPV Classification: Carcinogenic Risk

• Over 100 HPV strains identified

• Risk assessment based on transformative potential of a strain’s E proteins

• Low found in benign lesions only

• Intermediate found in benign lesions & invasive cancers

• High usually found in carcinomas; occasionally seen in benign lesions

Low Risk 60, 11, 42, 43, 44

Intermediate Risk

31, 33, 35, 51, 52, 58

High Risk 16, 18, 45, 56Furumoto et al., 2002.

Early Carcinoma Advanced Carcinoma

HPV 16

HPV 18

HPV 6

HPV 11

Cancer causing Types1,2,4 Non-cancer causing types1,2

• >75% of Cervical Cancer5,6

• ~50% of Vaginal & Vulvar Cancer590% of Anogenital warts5

HPV is a necessary cause of cervical cancer - 99.7%4

HPV

1.Schiffman M, Castle PE. Arch Pathol Lab Med. 2003;127:930–934. 2. Wiley DJ, Douglas J, Beutner K, et al. Clin Infect Dis. 2002;35(suppl 2):S210–S224. 3. Muñoz N, Bosch FX, Castellsagué X, et al. Int J Cancer. 2004;111:278–285. Reprinted from J Virol. 1994;68:4503–4505 with permission from the American Society for Microbiology Journals Department. 4. Walboomers JM, Jacobs MV, Manos MM, et al. J Pathol. 1999;189:12–19. 5. X. Castellsagué, S. de Sanjose, T. Aguado, K. S. Louie, L. Bruni, J.Muñoz, M. Diaz, K. Irwin, M. Gacic, O. Beauvais, G. Albero, E. Ferrer, S. Byrne,F. X. Bosch. HPV and Cervical Cancer in the World. 2007 Report. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Available at: www.who.int/hpvcentre6. Bhatla N et al.Vaccine (2008;26; 2811-17

Human Papillomavirus (HPV)

Human Papilloma Virus (HPV)

HPV Genome

• E1-E7 = “Early” genes (nonstructural)

• L1, L2 = Capsid genes• URR = upstream regulatory

region

• E6 & E7 proteins play major role in immortality & malignant transformation of infected cells

• E5 has role, but not required to maintain cancer phenotypeMunoz et al. 2006.

Early Genes Hijack Cell Cycle Checkpoint

• HPV’s E6 & E7 proteins interact with key cell cycle proteins including pRB & p53, effectively over-riding the G1/S-phase checkpoint

Mechanism1. E7 binds & phosphorylates pRB, activating E2F

transcription factor2. DNA replication proteins of host cell are then expressed;

unchecked S-phase occurs3. E6 marks p53 for proteolytic degradation so it cannot

activate apoptosis (note: absence of p53 is not necessary for E6 to cause immortalization)

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Common HPV Types and their effects

HPV Types Lead to:

Low-Risk

High-Risk

HPV 6, 11, 40,, 42, 43, 44, 54, 61, 70, 72, 81

HPV 16, 18,31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82

Benign cervical changes

Genital warts

Precancer cervical changes

Cervical cancer

Anal and other cancers

1. Cox. Baillière’s Clin Obstet Gynaecol. 1995;9:1. 2. Munoz et al. N Engl J Med. 2003;348:518.

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Human Papillomavirus

Cancer of cervix 100%

Cancer of esophagus .

Cancer of skin .

Cancer of X,Y,Z…. .

Cancer of mouth 3%

Cancer of throat 12%

Cancer of penis 40%

Cancer of vulva, vagina 40%

Cancer of anus 90%

Parkin DM et al. CA Cancer J Clin 2005; 55:74-108.

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Co-factors for HPV Infection

•Smoking•HIV infection •Other immune system defect•Pregnancy•Oral contraceptive use

Ferris et al. Modern Colposcopy. 2004.

HPV Infection

Low Grade

Lesions

High Grade

Lesions

Invasive Cancer

0–1 Year 0–5 Years 1–20 Years

HPV Infection may clear

Adapted from Pinto AP et al. Clin Obstet Gynecol. 2000;43:352–362.

Facts about HPV Infection

Facts about HPV Infection

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HPV Infections: Summary

• HPV can lead to genital warts• Persistent high-risk HP Most people are infected by HPV at some time• Immune system usually clears HPV, but not always• Persistent low-risk V can lead to pre-cancer

HPV

Long persistence of HPV can lead to cancer

Incidence (Women: all ages) - Cervical Cancer

1. X. Castellsagué, S. de Sanjose, T. Aguado, K. S. Louie, L. Bruni, J.Muñoz, M. Diaz, K. Irwin, M. Gacic, O. Beauvais, G. Albero, E. Ferrer, S. Byrne, F. X. Bosch. HPV and Cervical Cancer in the World. 2007 Report. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Available at: www.who.int/hpvcentre

1. X. Castellsagué, S. de Sanjose, T. Aguado, K. S. Louie, L. Bruni, J.Muñoz, M. Diaz, K. Irwin, M. Gacic, O. Beauvais, G. Albero, E. Ferrer, S. Byrne, F. X. Bosch. HPV and Cervical Cancer in the World. 2007 Report. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Available at: www.who.int/hpvcentre

Mortality (Women: all ages) - Cervical Cancer

Age Specific Incidence vs Mortality

2. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Summary report on HPV and cervical cancer statistics in India. 2007. [Accessed on 18th March 2008. Available at www. who. int/ hpvcentre c WHO/ICO Information Centre on HPV and Cervical Cancer

HPV Type Distribution - Invasive cervical cancer

1. X. Castellsagué, S. de Sanjose, T. Aguado, K. S. Louie, L. Bruni, J.Muñoz, M. Diaz, K. Irwin, M. Gacic, O. Beauvais, G. Albero, E. Ferrer, S. Byrne, F. X. Bosch. HPV and Cervical Cancer in the World. 2007 Report. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Available at: www.who.int/hpvcentre

HPV 16+18=76.7

%

HPV types and cervical cancer

1. Bosch FX et al. Vaccine 2008; 26S: K1–16. 2. Bhatla N et al. Vaccine 2008; 26(23):2811-2817.

Five most frequent and aggressive HPV types that cause cervical cancer worldwide

+ + +

HPV 16 HPV 18 HPV 45 HPV 31 HPV 33

+

These 5 HPV types are responsible for up to 92% of Cervical Cancer in India2

Years of Life Lost to Cervical Cancer*

*In women in the United States (2003), 1. Ries LAG, Harkins D, Krapcho M, et al. (eds). SEER Cancer Statistics Review, 1975–2003, National Cancer Institute. Bethesda, MD; 2006.

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19

18

Largest single cause of years of life lost to cancer in the developing world

Diagnosis

• 3% Acetic acid application• Lugol’s Iodine application• PAP smear• Liquid Based cytology• PCR• RT PCR• Multiplex PCR

Women were examined visually by simple speculum and colposcopically after application of 3 % acetic acid to cervix.

Equal detection rates of cervical abnormalities by both techniques.

WHEN LUGOL’S IODINE IS APPLIED TO THE CERVIX, THE NORMAL CELLS CONTAINING GLYCOGEN STAIN DARK BROWN. THE ABNORMAL CELLS ARE RAPIDLY DIVIDING AND ARE DEFICIENT IN GLYCOGEN HENCE, REMAIN UNSTAINED WHICH ARE FURTHER EVALUATED BY COLPOSCOPY & BIOPSY.

• Adenocarcinoma is difficult to detect with routine screening methods1

– The cervical smear brush cannot access the endocervical canal as easily as the outer surface of the cervix1

Adenocarcinoma is difficult to detect

Adenocarcinoma: may beinaccessible to the cervical

smear brush

Squamous cell carcinoma:usually accessible to the

cervical smear brush

Cervical smear brush

Cervix

Adenocarcinoma of the cervix- An Emerging concern

• Incidence increasing (20–25% of all cervical cancers), not prevented with traditional pap screening

• More aggressive and occurs in younger women

• > 90% of adenocarcinomas result from HPV 16, 18, 45, 33 and 311

• HPV 18 confers the highest risk

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Conventional Pap Smear

• Developed by Dr. George N. Papanicolaou in 1940’s

• Most common cancer screening test

• Key part of annual gynecologic examination

• Has greatly reduced cervical cancer mortality in U.S.

Ferris et al. Modern Colposcopy. 2004: 2-4, 49.Photo accessed from http://www.cytology-iac.org/Cytopaths/1998/cytoFall98.htm

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Screening with the Conventional Pap Smear

• Widely available• Inexpensive • But not perfect

– Screening test – not diagnostic– 7-10% of women need further evaluation– Low sensitivity – need regular repeats

Cervical Cytology Screening. ACOG Practice Bulletin No. 45. 2003; 102:417-27.

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New Liquid Pap Tests

• More accurate test– Thin, uniform layer of cells– Screening errors reduced by half

• Screening needed less often• Can test for HPV with same

specimen if abnormal cells found

• ExpensiveLinder J. et al. Arch Pathol Lab Med. 1998; 122: 139-144.

HPV-containing double stranded DNA

‘Empty’ non-infectious virus-like particle (VLP) mimics the

virus

Virus-like particles (VLPs) as HPV vaccine antigens mimic the virus structure

Stanley M, et al. Vaccine 2006; 24(suppl 3):S3/106–113.

Vaccines

• Gardasil (2006) : Contains HPV 16, 18, 6, 11• Cerverix (2009): Contains HPV 16, 18

– Females 10 through 25– For:

• Ca Cx• CIN• Adenocarcinoma in situ

– Gardasil for males too and for Ca Anus, warts etc

NexGen MoDxsEnd point PCR:• 1st Generation : PCR • 2nd Generation: Isothermal PCR

» NASBA» SDA

• 3rd Generation: Real Time PCR (21st Century):• Next Generation Multiplex Real Time PCR

• DPO technology• TOCE technology

DPO TM

Dual Primer Oligonucleotide

TOCE TM

Tagging Oligonucleotide Cleavage & Extension

• How to increase specificity with out changing the basic thermodynamics and kinetics of PCR?• Increased primer length increases specificity• BUT increased primer length increases the Tm

Principle of DPO™

NexGen MoDx Solutions

DPO TM

Dual Primer Oligonucleotide

TOCE TM

Tagging Oligonucleotide Cleavage & Extension

Principles of TOCE™

NexGen MoDx Solutions

Key Features and Benefits of DPO™/TOCE™

Key Features and Benefits of DPO™/TOCE™

What is the Paradigm Shift ?

Current Offering• Respiratory:

– RV 16– RB 5

• Genital– STI 7– HPV 28

• Each swab contains– Specimen collection

swab with a tip flocked with soft nylon fibre

– Polypropylene screw cap tube with 2 ml of eNAT transport medium

– Each swab has a molded breakpoint in the shaft

Sample collection & Transportation

• All sample must be collected using eNAT Swabs (COPAN)• Three different swabs are available

– eNAT Regular applicator (606CS01 R)– eNAT L Shaped Applicator (606CS01 L)– eNAT Pernasal applicator (606CS01 P)

• eNAT medium stabilizes & preserves RNA/DNA for prolonged time periods

• eNAT medium contains detergent & protein denaturant, so not suitable for culture based tests

• Transport at 5 to 25 C (in cold)

Collection of Cx Swab

• Each swab contains– Specimen collection

swab with a tip flocked with soft nylon fibre

– Polypropylene screw cap tube with 2 ml of eNAT transport medium

– Each swab has a molded breakpoint in the shaft

Current Offering• Respiratory:

– RV 16– RB 5

• Genital– STI 7– HPV 28