j.gras hpv mrna eurogin 2010

Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients

Jeremie M. Gras, M.D.

Clinical and Molecular laboratory

Cliniques Sud Luxembourg, Arlon, Belgium

bioMérieux Satellite Symposium

Eurogin Congress, Monte-Carlo, 18th February 2010


Plan

1. Background

2. Patients and methods

3. Results

4. Discussion

5. Conclusion

1. Background

• Cervical cancer is the second most common type of cancer occurring in women

• In 2005, cervical cancer was responsible for more than 250000 deaths

• Human Papillomavirus (HPV) is responsible for 99% of cervical cancer

Source: WHO

Cervical cancer

1. Background

• In the majority of cases, HPV infections regress spontaneously within two years1

• Only a small percentage progress to high grade lesions

• E6 and E7 proteins are viral oncogenes that are continuously expressed by malignant phenotypes

Detection of E6 and E7 mRNA provide more accurate prediction of cancer risk than DNA testing²

HPV mRNA test rationale

1Schiffman M, Castle PE, Jeronimo J et al. Lancet 2007; 370:890-907.

2Cattani P, Siddu A, D’Onghia S et al. J Clin Microbiol 2009; 47(7):2136-41.

1. Background

• HPV (16,18,31,33,45) E6 and E7 mRNA detection and typing using NASBA principle

• NASBA: Nucleic Acid Sequence Based Amplification³

EasyQ® HPV mRNA test

3 Compton J. Nature 1991; 350(6313): 91-2.

NASBA RT-PCR

Conditions Isothermic (41°C) Thermal cycles

Rapidity +++ +

Enzymes Reverse Transcriptase

RNAse H

T7 polymerase

Reverse Transcriptase

Taq Polymerase


Plan

1. Background


3. Results

4. Discussion

5. Conclusion

2. Patients and Methods

Lab description

• 600 files/ day, 24/7

• 40 % in patients; 60 % out patients (including GPs)

• Pre-analytic automation (since May 2009)

• Advanced QC solutions and protocols for clinical chemistry

• New infrastructure for molecular diagnostics (April 2008)

• ISO 15189 accredited for molecular biology (mandatory according to Belgian law)


ISO 15189 accreditation at Cliniques Sud Luxembourg

• 20 molecular tests have been accredited according to ISO 15189 norm

• Accreditation BELAC N° 370- MED, granted from 23/06/2009 to 22/06/2012

• HPV mRNA test is an accredited test in CSL Arlon Lab


Patients

• Study interval: from September 2008 to May 2009

• 332 HPV mRNA tests performed in 302 patients

• 30 tests were repeated for follow up of HPV disease

• Results will focus on the 302 primary HPV tests

• Mean age was 32.7 years old (15.8 to 83 years old)

• Cytology results:

2. Patients and MethodsMethods

1. Cytology first performed, then PreservCyt® vial was transmitted to our lab for preparation (40 mins)

2. Extraction on the easyMAG ® (40 mins)

3. Incubation (40 mins)

4. HPV mRNA amplification and detection using the EasyQ ® test (NASBA) (120 mins)

TAT for 24 samples: 4 hours


Plan

1. Background


3. Results

4. Discussion

5. Conclusion

3. Results

Overall Low rate of invalid results

3. Results

ASCUS

Negative; 66%

Invalid; 5%

HPV 16; 15%

HPV 18; 3%

HPV 31; 5%

HPV 45; 1%

HPV 33; 4%

HPV33-45; 1%

Negative

Invalid

HPV 16

HPV 18

HPV 31

HPV 33

HPV 45

HPV33-45

ASCUS: 66% have a negative HPV test

3. Results

L-SIL

Negative; 56%

Invalid; 4%HPV 16; 19%

HPV 18; 4%

HPV 31; 5%

HPV 45; 3%

HPV 33; 4%

HPV 16-31; 3%

HPV 16-33; 1%

HPV 18-31; 1%

Negative

Invalid

HPV 16

HPV 18

HPV 31

HPV 33

HPV 45

HPV 16-31

HPV 16-33

HPV 18-31

L-SIL: 56% have a negative HPV test

3. Results

H-SIL

HPV 1632%

HPV 18; 10%

HPV 315%

HPV 3310%

HPV 16-18; 10%

Negative; 7; 33%

HPV 16

HPV 18

HPV 31

HPV 33

HPV 16-18

Negative

HPV 58; 2 HPV 61; 1

HPV 51-81; 1

HPV 39; 1HPV 51; 2

HPV 39

HPV 51

HPV 58

HPV 61

HPV 51-81

Negative samples have been retested with a DNA method

N.B: HPV 61 and 81 are not HR HPVs

3. Results

Unclear cytology (N=48)

HPV 16; 4

HPV 18; 4

HPV 31; 1

Negative; 37

Invalid; 1

HPV 16-31; 1

HPV 16

HPV 18

HPV 31

HPV 16-31

Negative

Invalid

10 active infections with HR HPVs

3. Results

Specificity and NPV

• Calculated by comparison between HPV mRNA test and cytology results

• Specificity: 70.1 %

• Negative Predictive Value: 96.5 %

• Limitations: cervical cytology is a poor gold standard


Plan

1. Background


3. Results

4. Discussion

5. Conclusion

4. Discussion

• EasyQ® mRNA test gave a low number of invalid results (4%)

• Due to poor sample or bad RNA quality

• Re-testing allowed to find a valid result in most cases

Study findings (1/2)

a) Invalid results

• In patients with ASCUS, 66 % of HPV mRNA tests were negative

• In patients with L-SIL, 56 % of HPV mRNA tests were negative

• Overall, negative predictive value was 96.5 %

b) Results in ASCUS and L-SIL

4. Discussion

• 48 samples (17 %) had an unclear cytology screening results

• In several cases, another type of infection was suspected

• HPV mRNA test allowed to find 10 active infections with HR-HPVs

Study findings (2/2)

c) Interest in patients with unclear cytology results

• Patients with H-SIL are referred to colposcopy, irrespective of mRNA test result

• H-SIL samples with negative mRNA test were re-tested with a DNA test: in all cases, it was positive for HPV subtypes that technically can not detected by HPV EasyQ® mRNA test

d) Patients with H-SIL


Plan

1. Background


3. Results

4. Discussion

5. Conclusion

5. Conclusion

• Combination of easyMAG® automated extraction and EasyQ® mRNA test proved to be a fast and reliable method to detect and type HR HPVs in cervical samples

• It detects molecular markers that are closely related to cancer risk

• Integration of HPV mRNA detection in screening algorithms could reduce unneeded additional investigations in many patients with ASCUS or L-SIL

• It allows to detect active HPV infections in patients with unclear cytology results

Thanks

Molecular Biology Laboratory, Cliniques Sud Luxembourg ARLON, Belgium

Nicolas Hougardy Head Molecular Lab

Pierre Goffinet Lab Director

Brigitte LégerNathalie PinonBernadette Legère MLTsAndrée LemaireFrançoise SobletMaggy Conrardy

Thanks

And thank YOU for your attention !


Jeremie M. Gras, M.D.

Clinical and Molecular laboratory

Cliniques Sud Luxembourg, Arlon, Belgium

bioMérieux Satellite Symposium

Eurogin Congress, Monte-Carlo, 18th February 2010

j.gras hpv mrna eurogin 2010

Documents