title: Discrimination of grade 2 and 3 cervical intraepithelial neoplasia by means of analysis of water soluble proteins recovered from cervical biopsies.
abstract: BACKGROUND
  Cervical intraepithelial neoplasia (CIN) grades 2 and 3 are usually grouped and treated in the same way as "high grade", in spite of their different risk to cancer progression and spontaneous regression rates. CIN2-3 is usually diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. This procedure virtually eliminates the availability of water-soluble proteins which could have diagnostic and prognostic value.
  
  
  OBJECTIVE
  To investigate whether a water-soluble protein-saving biopsy processing method followed by a proteomic analysis of supernatant samples using LC-MS/MS (LTQ Orbitrap) can be used to distinguish between CIN2 and CIN3.
  
  
  METHODS
  Fresh cervical punch biopsies from 20 women were incubated in RPMI1640 medium for 24 hours at 4°C for protein extraction and subsequently subjected to standard FFPE processing. P16 and Ki67-supported histologic consensus review CIN grade (CIN2, n = 10, CIN3, n = 10) was assessed by independent gynecological pathologists. The biopsy supernatants were depleted of 7 high abundance proteins prior to uni-dimensional LC-MS/MS analysis for protein identifications.
  
  
  RESULTS
  The age of the patients ranged from 25-40 years (median 29.7), and mean protein concentration was 0.81 mg/ml (range 0.55 - 1.14). After application of multistep identification criteria, 114 proteins were identified, including proteins like vimentin, actin, transthyretin, apolipoprotein A-1, Heat Shock protein beta 1, vitamin D binding protein and different cytokeratins. The identified proteins are annotated to metabolic processes (36%), signal transduction (27%), cell cycle processes (15%) and trafficking/transport (9%). Using binary logistic regression, Cytokeratin 2 was found to have the strongest independent discriminatory power resulting in 90% overall correct classification.
  
  
  CONCLUSIONS
  114 proteins were identified in supernatants from fresh cervical biopsies and many differed between CIN2 and 3. Cytokeratin 2 is the strongest discriminator with 90% overall correct classifications.
authors.affiliation: Pathology Department, Stavanger University Hospital, Armauer Hansen Road 20, Stavanger, Norway. jpabaak@yahoo.com.
authors.forename: Kai-Erik
authors.initials: KE
authors.lastname: Uleberg
authors.forename: Ane Cecilie
authors.initials: AC
authors.lastname: Munk
authors.forename: Cato
authors.initials: C
authors.lastname: Brede
authors.forename: Einar
authors.initials: E
authors.lastname: Gudlaugsson
authors.forename: Bianca
authors.initials: B
authors.lastname: van Diermen
authors.forename: Ivar
authors.initials: I
authors.lastname: Skaland
authors.forename: Anais
authors.initials: A
authors.lastname: Malpica
authors.forename: Emiel Am
authors.initials: EA
authors.lastname: Janssen
authors.forename: Anne
authors.initials: A
authors.lastname: Hjelle
authors.forename: Jan Pa
authors.initials: JP
authors.lastname: Baak