J.Gras HPV mRNA Eurogin 2010
Transcript of J.Gras HPV mRNA Eurogin 2010
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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
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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
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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
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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
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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.
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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
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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
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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)
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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
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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:
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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
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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
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3. Results
Overall Low rate of invalid results
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Thanks
And thank YOU for your attention !
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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