Page 84 - Strategies for non-invasive managementof high-grade cervical intraepithelial neoplasia - prognostic biomarkers and immunotherapy Margot Maria Koeneman

Chapter 4
consensus scoring of three observers (EG, AM, IO), followed by independent quality control of a fourth observer (JB). All used the same microscope (40 × objective 0.52 mm, numerical aperture 0.65). In case of discrepancy of > 10%, which occurred in 8% of all cases, consensus recount was done. All women underwent a Loop Electrosurgical Excision Procedure (LEEP) after a median of 113 days follow-up (range 84-171 days). The natural history of the baseline cervical lesion during the follow-up period was evaluated in the LEEP specimen. Regression was defined as CIN1 or less in the LEEP specimen. Further details on histological evaluation, HPV genotyping and lesions size measurements can be found in the original article.[5] Out of this cohort, representative and sufficient baseline biopsy material for 3q26 analysis was available for 19 patients. These patients were included in the pilot study.
FISH procedure
FISH analysis was performed on the baseline biopsies. The 3q specific FISH was performed on 4 μm thick FFPE tissue sections fixed onto Superfrost Plus Microscope Slides (Thermo Fisher Scientific). The tissue sections were first heated for 15 min at 80°C, then dewaxed, hydrated and microwaved for 10 min at 100°C in a 10 mM Na-Citrate pH buffer and incubated at room temperature for 20 min to cool down. Subsequently, the sections were washed in demineralized water, rinsed in 0.01 M HCl and digested with 2.5 mg of pepsin in 0.01 N HCl and post-fixed in 1% formaldehyde in PBS for 5 min at room temperature. Subsequently, the 3 centromere probe (pα3.5) and 3q26 probe (3q26.1: BAC23 RP11-264D7, Map position 3q26.1 – 26.3 close to the TERC locus), were labeled with Digoxigenin (3c) and Biotin (3q) in a nick translation labelling (Jena Bioscience GmBH, Jena, Germany). The probes were hybridized at a concentration of 2 αg/μl (3c), 5 αg/μl (3q); 10 x excess COT, and 75x excess of carrier DNA (salmon sperm DNA) in 50% formamide; 2x SSC; 10% dextran sulphate. The probe was applied under a coverslip, simultaneously denatured for 10 min at 80°C and hybridized overnight at 37°C. After hybridization, the preparations were washed for 5 min at 61°C in a solution, containing 2 × SSC, 0.05% tween-20 (Janssen Chimica, Beerse, Belgium) and 0.1 × SSC (the washing was carried out twice). The hybridized FISH probe was detected with a triple layer detection method, consisting of 1. FITC-conjugated avidin (Av-FITC, 1:100 dilution, Vector Laboratories) / Monoclonal anti-Digoxigenin (MαDig, 1:100 dilution, Sigma, USA, St Louis MO); 2. Botinylated Goat anti-Avidin (Bio-GαA, 1:100 dilution, Vector Laboratories USA) / Rabbit anti Mouse-TRITC (1:100 dilution, Dako, Glostrup, Denmark) and 3. Av-FITC / Swine anti Rabbit- TRITC (1:100 dilutionDako). Finally, the slides were washed in PBS containing 0.05% Tween-20, dehydrated in an ascending ethanol series and mounted in Vectashield (Vector Laboratories), containing DAPI (Sigma: 0.5 μg/μl). Images were recorded with the Metasystems Image Pro System (black and white CCD camera; Sandhausen, Germany), mounted on top of a Leica DM-RE fluorescence microscope.[15]
FISH evaluation
The FISH signals were interpreted by two analysts (MU, AH), who were blinded to the outcome data. Dysplastic areas were identified based on p16 staining and were scanned for FITC and TRITC signal copy numbers. The copy number was estimated as previously described to detect disomy, tetrasomy up to nonasomy by means of the determination of the maximum copy number and heterogeneity in formalin fixed and paraffin embedded tissue sections. The validity of this
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