Σταύρος Π. Δέρδας M.D.,M.Sc.,Ph.D.

• Cervical smears were obtained from a consecutivesample of 80 patients, who attended 401 MilitaryHospital of Athens , in order to have their annualgynaecological check-up.

• The majority of our patients were Caucasian withan age range of 18-51 years (mean age=29.17years).

• The patients were also offered HPV test with theknowledge that it is not part of the screening.Ethical approval was granted by the EthicsCommittee of 401 Military Hospital of Athens andall participants provided written, informed consent.

• The classification of cytological findings wasperformed according to the criteria of the Bethesda2001 system and were categorized into normal,atypical squamous cells of unknown significance(ASCUS), low-grade squamous intraepitheliallesions (LSIL) and high-grade squamousintraepithelial lesions (HSIL).

Procedure used to collect cells from the cervixso that they use a small sampler — a tinyspatula or brush — to gently collect cells fromyour cervix. The cells are sent to a lab to betested.

Liquid based cytology (LBC), singlecollection devices and stained/coverslipslides.

• Cervical cell scrapings were collected and preserved in Thin Prep (PreservCytSolution Corporate Headquarters: Hologic, Inc. Ltd., UK).

• Five millilitres were used for DNA extraction. They were centrifuged and dilutedinto lysis buffer QIAampDNAMini Kit.

• The samples were then subjected for total nucleic acid extraction, according to themanufacturer’s instructions. Nucleic acids were eluted in 55 μl of elution buffer.

• Aliquots were stored appropriately for further processing.

• Quantity and quality of extracted DNA was checked by spectrophotometer(Nanodrop®) as well as by running on 0.8% agarose gel.

• After validating the DNA extraction results, DNA was stored at −20°C until PCR wasperformed.

• Liquid based cytology (LBC) slideswere prepared according toprotocols from the manufacturer(CellSolutions, LLC).

• Scoring of immunocytochemistryresults were performed on thebasis of the staining intensity intofour grades (0 to +++)

A B C

D E F

G H I

Liquid based cytology (LBC) showing, (Α) Koilocytes(Pap, X 400), (B) LGSIL (Pap, X 100), (C) HGSIL (Pap, X400), (D) LGSIL (Pap, X 400), (E) HGSIL (Pap, X 400), (F)Normal squamous cells (Pap, X 100), (G) Cervico-vaginitis (Pap, X 100), (H) Atrophy (Pap, X 100), (I) Fungi(Pap, X 400).

• The cobas® HPV tests are automated qualitativein vitro tests for the detection of humanpapillomavirus (HPV) DNA in patient specimens.

• The tests utilize amplification of target DNA bythe Polymerase Chain Reaction (PCR) andnucleic acid hybridization for the detection of 14high-risk HPV (hrHPV) types in a single analysis.

• Internal control: The ß-globin internal cellularcontrol helps prevent false negatives. HPVnegative specimens with a negative ß-globinresult are flagged as invalid, helping to preventreporting of false negative results

• Use of AmpErase Enzyme: Each reactioncontains AmpErase Enzyme, reducing the risk offalse positive results from carry-overcontamination by differentiating amplificationproducts from target molecules

❖ Assays are validated in clinical performancestudies (e.g. cobas® 4800 HPV test was validatedin the ATHENA Trial)

❖ Validated for detection of >CIN2 lesions and notsimply presence of HPV

❖ Validated to the standards set forth ininternational guidelines for HPV testing forcervical screening purposes

• The isothermic gene amplificationmethod ‘Nucleic Acid SequenceBased Amplification’ (NASBA) hasmany features, which makes itwellsuited for use with RNAtargets.

• The method was performed inreal-time format, using thePreTect HPV-Proofer kit to detectfull-length mRNA from only HPV16, 18, 31, 33 and 45 (NorChipAS, Oslo, Norway).

❖ A detailed description of the HPV-Proofer protocols has beenpublished.

HPV type

Assay 16 18 31 33 45 Real-time PCR 2 - 1 1 1

E6/E7 mRNA 1 - 1 - -

TABLE I. Correlations between HR-HPV types foundby using real-time PCR and oncogene mRNAdetection.

CIN grade(%)

Assay NA I II/III

Real-time PCR 10/22(45.5) 2/22(9.1) 5/22(22.7)

E6/E7 mRNA 1/10(10) - 1/5(20) NA, no atypia. A total of 22 cases were scored in real-time PCR.

TABLE II. Distribution of percentage positive casesin real-time PCR and E6/E7 mRNA assays in differentgrades of CIN.

362321

0

10

20

30

40

80 women with cytologicalsigns

LSIL

HSIL

ASCUS

2

5

10

31

CIN I

CIN II/III

ASCUS

WNL

UHD

8,33%

20,83%

41,6%

12,5%

4,16%

Chart II. Various degrees of CIN according to histopathology.

Chart I. Cytological signs of atypia.

*Within Normal Limits

*Unclear Histological Diagnosis

• Οι θετικές περιπτώσεις HR-HPV ήταν επίσης θετικές στην ανάλυση mRNA. Αυτό ήταν αναμενόμενο,δεδομένου ότι δεν είναι όλες οι περιπτώσεις μολυσμένων από HR-HPV μεταγραφικά δραστικές γιαέκφραση Ε6 / Ε7.

• Είναι ενδιαφέρον να σημειωθεί ότι η πολύ υψηλή θετικότητα (71%) για HR-HPV στις περιπτώσεις WNLείναι σε αντίθεση με τα χαμηλά ποσοστά στην ανάλυση Ε6 / Ε7 mRNA (14%).

• Η διαπίστωση ότι τα περιστατικά CIN I και Εντός των Περιορισμένων Ορίων (Within Normal Limits )υποδεικνύουν την ογκογόνο έκφραση HR-HPV μπορεί να είναι ένα μήνυμα ότι αυτοί οι ασθενείς έχουναυξημένο δυνητικό κίνδυνο για την ανάπτυξη βλαβών υψηλού βαθμού.

• Στα δείγματα που βαθμολογήθηκαν ως CIN II βρήκαμε HR-HPV E6 / E7 mRNA μόνο σε 20% (1/5) τωνπεριπτώσεων.

• Κανένα δείγμα, το οποίο ήταν αρνητικό με real-time PCR, δεν ήταν θετικό στο σύστημα ανίχνευσηςmRNA Ε6 / Ε7.

• Prof. D. A. Spandidos

• Dr. Spyros Gerou M.D., Ph.D.• Elpida Papadopoulou B.Sc.• Vicki Pavlidou B.Sc.• Korina Kydonopoulou M.Sc.• Marios Kampouris Ph.D.

• Dr. Stavros Archondakis M.D., Ph.D