Viral and Host Factors in Vulvar Disease DR MICHELLE ETHERSON 26 TH OF APRIL 2016

Viral and Host Factors in Vulvar Disease DR MICHELLE ETHERSON 26 TH OF APRIL 2016

Human Papillomavirus (HPV) Small non-enveloped Double stranded DNA Virus Genome 8000 basepairs 8 coding genes, Early (E) and Late (L) and a noncoding long control region (LCR) Doorbar et al (2005)

HPV Classification Similarity between the DNA sequence at each virus s most conserved region of genome, L1 Open Reading Frame (ORF) devilliers et al 2004

HPV Risk of Malignancy Alpha-papillomavirus most important in anogenital disease Cervical, Anal, Penile, Vaginal, Vulvar and their preinvasive intraepithelial stages Different HPV types confer different oncogenic risk Low Risk types include HPV 6 and 11 High Risk types include HPV 16, 18, 31, 33 HPV 16 provides the greatest carcinogenic risk

HPV 16 Major variant lineages A(1-4) = European(E) and Asian(As) lineages B(1-2) = African-1(Af-1) lineages C = African-2(Af-2) lineages D(1-3) = Asian-American(AA) lineages Cervical disease - variable risk of persistence and oncogenesis Non-European lineages (B/C/D) more pathogenic Increased risk of persistence - T350G (within E6 gene) in European lineages Burk et al (2013)

Vulvar Intraepithelial Neoplasia (VIN) and Vulvar Cancer (VSCC) Constructed using ISD data from http://www.isdscotland.org/health-topics/cancer/cancer- Statistics/Female-Genital-Organ/#vulva ICD-51 Vulvar Cancer

Preti et al (2014)

Role of HPV in VIN and VSCC Worldwide prevalence of HPV in vulvar disease 80% in VIN 40% in VSCC HPV 16 predominates Risk of progression between 9-16% in untreated and 3% in treated higher risk in dvin Recurrence - one third of patients Treatment morbid Prophylactic Vaccination

Could host genetic variation contribute?

Single Nucleotide Polymorphisms (SNPs) Single base pair change in a genetic sequence present in at least 1% of the population SNPs can affect gene regulation and protein expression Infectious diseases and response to treatment, cancer susceptibility and prognosis

CXCR1 Work within our Group Association of CXCR1 SNP with CIN Interleukin-8 Receptor A Chemotaxis Tumorigenesis IL-8 + CXCR1 increased in cancer including cervical Disease progression

CXCR1 SNP rs2234671 Non-synonymous SNP G C Serine May to Threonine amino acid at position 276 alter structure and binding

CXCR1 SNP Associations Increased risk:- Asymptomatic bacteriuria in women and acute pyelonephritis in children Mucosal Leishmaniasis Active Hepatitis B and Cirrhosis Lung cancer and Gallbladder cancer Poorer response to bevacizumab and oxaliplatin-based chemotherapy in metastatic colon cancer

Specific Research Questions Identify viral and genetic factors contributing to VIN and VSCC development and progression This will provide valuable information for both primary prevention and improving clinical management What is the prevalence of HPV genotypes and HPV 16 variants in VIN and VSCC samples? Is the CXCR1 SNP rs2234671 associated with VIN and VSCC?? Is the CXCR1 SNP rs2234671 functional?

HPV 16 Variants Analysis of HPV genotypes and HPV 16 variants Collaborative work with Katie Wakeham 240 extracts of nucleic acids samples from FFPE VIN and VSCC cases HPV Genotyping via luminex based assay Clinical database produced

VIN Cases VSCC Cases Average Age (all) 49.8 60 Average Age (HPV related) 47.5 52 Total Number 166 66 % HPV Positive 78.3% (130) 34.8% (23) % HPV 16 Mono-Infection 81.5% (106) 91.3% (21) % Other or Mixed Infection 18.5% 8.7%

VIN HPV 16 106 HPV 33 9 HPV 18 3 HPV 42 2 HPV 45 1 HPV 16, 33 2 HPV 16, 45 1 HPV 16, 18 1 HPV 6, 18 1 HPV 16, 42 1 HPV 16, 45 1 HPV 16, 33, 53 1 HPV 6, 11, 16 1 VSCC HPV 16 21 HPV 31 1 HPV 16, 42 1

Variant Analysis Sanger sequencing following PCR amplification of 2 regions within E6 Swan et al 2006

Primer set 1 E6(75) Forward and E6(206) Reverse 1000 bp 500 bp 200 bp Expected product size 131 100 bp 1000 bp 500 bp 200 bp 8 3 0 7 8 3 0 8 8 3 1 0 8 3 1 3 8 3 1 7 8 3 2 0 8 3 2 4 8 3 2 7 S i H a H e L a W a t e r Expected product size 114 100 bp Primer set 2 E6(274) Forward and E6(388) Reverse If bands present for both E6 regions PCR products purified and sent for sequencing

Sequence file and Chromatograph Further analysis of chromatograph where N present in BioEdit and corrected if possible

Sequence Analysis Identified variants using pairwise alignment and MView tools Sequences compared to the reference genome (k02718.1) and published variant sequences Phylogenetic analysis and clades defined Test associations of variants with disease outcomes

Nucleotide Position - Substitution Group 109 131 132 135 143 145 178 350 No of VIN Cases No. of VSCC cases Reference T A G A C G T T 43 10 E350G....... G 28 4 E135C... C.... 1 0 E131G. G..... G 3 0 E131G.2. G...... 1 0 E109C C...... G 2 1 As-178G...... G. 2 1 Af-2 C. T. G T.. 0 1

E109 C (A1) E350 G (A1) 32 cases E131G (A2) E131G.2 E135 C European Phenotype/Reference (A1) 53 Cases Af-2 (C) As-178G (A4 or European Asian)

SNP analysis All vulvar samples SNP genotyped using real time PCR and meltcurve analysis Primer/probes designed to amplify SNP rs2234671 Different alleles have different melting temperatures Melting peak profile determines genotype

Homozygous G/G Homozygous C/C Heterozygous G/C Water

G/G

G/G

Nil

G/G

G/C

G/G

HPV Negative Disease Allele Frequency n G C Controls 178 93% 7% VIN+VSCC 77 92% 8% VIN 33 91% 9% VSCC 37 93% 7% HPV Positive Disease Allele Frequency n G C Controls 84 93% 7% VIN+VSCC 158 90% 10% VIN 130 91% 9% VSCC 23 80% 20%

Is the C allele associated with increased risk of HPV positive VSCC?

G C HeLa transfected with GFP plasmid HeLa transfected with G Allele plasmid HeLa transfected with C Allele plasmid

Summary HPV important casual factor in VIN and VSCC Viral variants and Host SNPs may play a role in disease development and outcome Paucity of knowledge within vulvar disease First Scottish study regarding HPV genotype and variant prevalence Similar HPV prevalence compared to worldwide data Provides important information when considering prophylactic vaccination strategies CXCR1 SNP Interesting! Identify host and viral factors to enable patient stratification and enhance clinical management

Acknowledgements Dr Kate Cuschieri Professor Simon Herrington Professor Juergen Haas Professor Tim Aitman Dr Ramya Bhatia Professor Mark Arends Professor Sarah Howie Professor David Harrison Dr Katie Wakeham Dr Richard Weller Duanduan Cong Itziar Serrano Catherine Moore Daniel Guerendiain Norman Stenhouse

Functional Work Site Directed Mutagenesis Plasmid with CXCR1 gene with C allele and G allele produced HEK293 Cells Transfected with plasmids Chemotaxis assay

Negative Control HEK293 cells No Transfection Positive Control MM-6 cells HEK293- transfected with C allele plasmid HEK293- transfected with G allele plasmid