j.gras hpv mrna eurogin 2010
TRANSCRIPT
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
Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients
Plan
1. Background
2. Patients and methods
3. Results
4. Discussion
5. Conclusion
Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients
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
Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients
Plan
1. Background
2. Patients and methods
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)
2. Patients and Methods
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
2. Patients and Methods
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
Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients
Plan
1. Background
2. Patients and methods
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
Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients
Plan
1. Background
2. Patients and methods
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
Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients
Plan
1. Background
2. Patients and methods
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 !
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