Atypical Squamous Cells of Undetermined Significance

Six NHS laboratories had partially converted to primary hrHPV testing between May and August 2013. These laboratories in Bristol, Liverpool, Manchester, Norwich, Northwick Park (West London), and Sheffield represent approximately 13% of the Cervical Screening Programme. Conversion, which involved around one third of screening samples, was population based. Distribution of primary hrHPV testing was based on clusters of general practices and was not subject to random allocation. Rather, allocation to liquid based cytology or hrHPV testing was a consequence of practical considerations such as maintaining one management protocol for each colposcopy unit, whereby colposcopy units often serve defined administrative clusters. Samples from women who were screened after a routine invitation were collected in primary care in either ThinPrep (Hologic, Marlborough, MA) or SurePath (Beckton Dickinson, Sparks, MD) liquid based cytology media, regardless of whether liquid based cytology or hrHPV was the primary screening test. hrHPV testing was performed with either the Cobas 4800 (Roche, Rotkreuz, Switzerland, or Branchburg, NJ), RealTime (Abbott, Wiesbaden, Germany), APTIMA (Hologic, Manchester, UK), or, to a limited degree, Hybrid Capture 2 (Qiagen, Gaithersburg, MD) assays. Each laboratory used a unique combination of liquid based cytology medium and hrHPV assay. All screening tests have been approved for use in the English Cervical Screening Programme following the official validation protocols.
The pilot adhered to the nationally recommended age range (25-64) and screening intervals (three years for women aged <50 and five years thereafter) for both screening tests. In women screened with hrHPV testing, cytology was not blinded to the hrHPV test result. Women were immediately referred for a colposcopy if their hrHPV test was positive and cytology showed any grade of abnormality. Women who were hrHPV positive with negative cytology were recommended for early recall at 12 months, at which point they were referred for a colposcopy if they remained persistently hrHPV positive and had developed any cytological abnormalities. Three laboratories referred women with persistently negative cytology at 12 months if their samples showed persistent infection with HPV 16/18. Other women who were hrHPV positive and cytology negative were offered further early recall at 24 months, and were referred for a colposcopy if they showed a persistent hrHPV infection regardless of their cytology. Women screened with cytology were recommended for a colposcopy if their cytology showed high grade abnormalities consistent with high grade squamous intraepithelial lesions in Bethesda 2001 terminology or low grade abnormalities consistent with either atypical squamous cells of undetermined significance (ASCUS) or low grade squamous intraepithelial lesions combined with a positive reflex test for hrHPV. Cytological glandular abnormalities were classified among high grade squamous intraepithelial lesions. Women not referred to colposcopy or early recall were routinely recalled at three or five years.
Screening was conducted in primary care, and training in relation to hrHPV based screening was provided. The laboratories routinely monitored compliance with referral for a colposcopy, and there was a safety net in place for women who did not attend. Colposcopy was conducted according to national clinical practice guidelines. All laboratories and colposcopy clinics took part in the national quality assurance programmes.

HPV‐positive and/or Pap‐abnormal women were followed up regularly at a 3‐month interval for 2 years. Women with HPV infections or cytologically diagnosed with ASC‐US or worse lesions (i.e., ≥ASC‐US) were referred to colposcopy before initiating a regular follow‐up schedule. If their biopsy results indicated an intraepithelial squamous lesion lower than HSIL (e.g., cervicitis, LSIL), the women were then involved in one of the three follow‐up cohorts, namely, HPV‐positive Pap‐normal, Pap‐abnormal biopsy‐negative and biopsy‐confirmed LSIL, based on their initial HPV, cytological and biopsy diagnoses. During follow‐up, the women were offered regular HPV DNA and Pap tests. If their regular Pap results revealed a persistent LSIL lesion over 1 year or a lesion worse than their initial cytological/biopsy diagnoses, the women were referred to colposcopy again. For women whose HPV DNA test revealed evidence of an infection with a novel viral genotype(s) or a relapse of former viral infection following an intermittent negative viral DNA test, colposcopy and biopsy (if necessary) were also referred. The (primary) endpoint event was biopsy‐confirmed HSIL or invasive cervical cancer within 2 years of follow‐up.

For analysis of the meiotic product, we crossed a MATa with a MATalpha haploid strain, selected for diploids based on auxotrophy or antibiotic resistance, and patched the diploid strain on rich pre-Sporulation plates (YP agar with 6% [w/v] glucose]. Then we froze part of the patch and transferred part of the patch into Sporulation medium (1% potassium acetate, 0.005% zinc acetate buffer) and incubated the cultures with shaking at 23 °C. After a few days, the sporulation cultures were treated in a ratio of 1:1 [v/v] with Lyticase (L4020 Sigma Aldrich, 2.5 mg/ml, 200 units/µl, in 1 M D-Sorbitol) to digest the ascus. After 15–20 min at room temperature, the culture was applied to an agar plate and tetrads were dissected using a Singer micromanipulator. Colonies of haploid spores grew at 30 °C for three days. Images were taken at 48 and 72 h with the ChemiDoc™ Touch Imaging System (Bio-Rad). After three days, the spores were replica plated, genotypes were scored and strains were frozen in 15% glycerol-containing cryopreserved stocks at −80 °C. Strains are listed in Supplementary Data 2.