Page 70 - Strategies for non-invasive managementof high-grade cervical intraepithelial neoplasia - prognostic biomarkers and immunotherapy Margot Maria Koeneman

Chapter 3
Study design and patient population
The patient population was extracted from a prospective population based cohort study, conducted at the Stavanger University Hospital, Norway.[2] The original cohort consisted of 162 women aged 25–41 years, with histologically proven CIN 2 or CIN 3 lesions. The lesions were diagnosed in diagnostic biopsies at baseline. 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. Histological evaluation was performed on standard Hematoxylin Erythrosin Saffran (HES) sections, according to the World Health Organization criteria. Ki67 and p16 were used to support their diagnoses. HPV genotyping was performed by Linear Array (Roche) on DNA- material from the formalin fixed paraffin embedded cervical biopsy at inclusion. Details on histological evaluation and HPV genotyping can be found in the original article.[5] From this cohort, 34 patients were included for analysis in the current study, based on the availability of sufficient blood samples for HLA analysis. EDTA blood was sampled at the same time as the biopsy and stored in a biobank.
DNA isolation
DNA isolation was performed by qualified staff with expertise in molecular biology in a Molecular Laboratory at the Pathology Department of Stavanger University Hospital, Stavanger, Norway. Until use the DNA had been stored in Biosphere safe seal Eppendorf tubes, (Sarstedt Inc., Thermo Fisher Scientific, MA, US) in a −80°C biobank freezer. The EZNA ® Tissue kit (Omega bio- tek, Norcross GA, USA) was used for purification of DNA from 250 μl frozen whole blood diluted (4.429 mmol/l) with the anticoagulant Ethylene Diamine Tetraacetic Acid (EDTA). The protocol for Blood and body fluids was followed according to the manufacturer`s instructions. DNA was purified using the HiBind ® DNA Mini Column and eluted in 50μl Elution Buffer.
HLA procedure
The HLA procedure was performed at the Tissue Typing Laboratory of the Department of Transplantation Immunology, Maastricht University Medical Centre, Maastricht, The Netherlands. The tissue typing laboratory has been accredited by EFI. HLA class I and class II typing was performed using the Luminex (Austin, TX, USA) sequence specific oligonucleotide (SSO) hybridization method. The SSO kits (One Lambda, Los Angeles, CA, USA) were used according to the manufacturer’s guidelines.[13] DNA amplification and hybridization were automated using the LabXpress (One Lambda). The fluorescence values were read by a Luminex Labscan and HLA typing assignments were obtained using HLA Fusion 3.0 software (One Lambda). All data have been generated simultaneously (i.e. using the same reagents, conditions and allele assignment databases (IPD-IMGT/HLA)). Blinding was not applicable, as HLA typing is not subject to interpretation.
Outcome measures and criteria for biomarker performance
The outcome measures were defined as (1) the correlation between HLA alleles and spontaneous regression of high-grade CIN and (2) the correlation between HLA alleles and HPV genotype in high-grade CIN lesions. No previous biomarker performance values are available for HLA types.
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