Search

Aim: T-helper cells could play an important role in the pathogenesis of chronic lymphocytic leukemia (CLL), a common B-cell neoplasm. Although CLL cells can present soluble antigens targeted from the B-cell receptor to T-helper cells via major histocompatibility complex (MHC) class II, antigens recognized by some CLL cells may be encountered in a particulate form. Here the ability of CLL cells to internalize and present anti-immunoglobulin M (IgM) beads as a model for the interaction of CLL cells with particulate antigens was investigated. Methods: The effect of anti-IgM beads on antigen presentation pathways was analyzed using RNA-seq and internalization of anti-IgM beads by primary CLL cells was investigated using confocal microscopy and flow cytometry. Antigen presentation was investigated by analyzing activation of a T-cell line expressing a T-cell receptor specific for a peptide derived from mouse κ light chains after incubating CLL cells with a mouse κ light chain-containing anti-IgM monoclonal antibody. Kinase inhibitors were used to characterize the pathways mediating internalization and antigen presentation. Results: Stimulation of surface IgM of CLL cells increased expression of the antigen presentation machinery and CLL cells were able to phagocytose anti-IgM beads. Internalization of anti-IgM beads was associated with MHC class II-restricted activation of cognate T-helper cells. Antigen presentation by CLL cells was dependent on activity of spleen tyrosine kinase (SYK) and phosphatidylinositol 3-kinase delta (PI3Kδ) but was unaffected by inhibitors of Bruton’s tyrosine kinase (BTK). Conclusions: CLL cells can internalize and present antigen from anti-IgM beads. This capacity of CLL cells may be particularly important for recruitment of T-cell help in vivo in response to particulate antigens.

Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma–associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell–like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell–like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell–specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN–expressing M2-polarized macrophages.

Tumour cells from patients with chronic lymphocytic leukaemia (CLL) can express unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain (IGHV) genes with differing clinical behaviours, variable B cell receptor (BCR) signalling capacity and distinct transcriptional profiles. As it remains unclear to what extent these differences reflect the tumour cells’ innate pre/post germinal centre origin or their BCR signalling competence, we applied RNA sequencing, small RNA sequencing and DNA methylation array analysis to 38 CLL cases categorised into three groups by IGHV mutational status and BCR signalling capacity. We identified 492 mRNAs and 38 miRNAs differentially expressed between U-CLL and M-CLL, but only 9 mRNAs and 0 miRNAs associated with BCR competence within M-CLL. A significant proportion of the IGHV-associated miRNAs derived from chr14q32 clusters (14/38 (37%)), where all miRNAs were co-expressed with the MEG3 lncRNA, as part of the DLK1-DIO3 genomic imprinted region, a locus of known importance in the pathogenesis of other human tumours. Integrative in silico analysis of miRNA/mRNA data revealed pronounced regulatory potential for the 14q32 miRNAs, potentially accounting for up to 25% of the IGHV-related transcriptome signature. GAB1, a positive regulator of BCR signalling, was predicted to be regulated by five 14q32 miRNAs and we confirmed that two of these (miR-409-3p and miR-411-3p) significantly repressed activity of the GAB1 3’UTR. Our analysis demonstrates a potential key role of the 14q32 miRNA locus in the regulation of CLL-related gene regulation.

In follicular lymphoma (FL), surface immunoglobulin (sIg) carries mandatory N-glycosylation sites in the variable regions, inserted during somatic hypermutation. These glycosylation sites are tumor-specific, indicating a critical function in FL. Added glycan unexpectedly terminates at high mannose (Mann) and confers capability for sIg-mediated interaction with local macrophage-expressed DC-SIGN lectin resulting in low-level activation of upstream B-cell receptor signaling responses. Here we show that despite being of low-level, DC-SIGN induces a similar downstream transcriptional response to anti-IgM in primary FL cells, characterized by activation of pathways associated with B-cell survival, proliferation and cell–cell communication. Lectin binding was also able to engage post-transcriptional receptor cross-talk pathways since, like anti-IgM, DC-SIGN down-modulated cell surface expression of CXCR4. Importantly, pre-exposure of a FL-derived cell line expressing sIgM-Mann or primary FL cells to DC-SIGN, which does not block anti-IgM binding, reversibly paralyzed the subsequent Ca2+ response to anti-IgM. These novel findings indicate that modulation of sIg function occurs in FL via lectin binding to acquired mannoses. The B-cell receptor alternative engagement described here provides two advantages to lymphoma cells: (i) activation of signaling, which, albeit of low-level, is sufficient to trigger canonical lymphoma-promoting responses, and (ii) protection from exogenous antigen by paralyzing anti-IgM-induced signaling. Blockade of this alternative engagement could offer a new therapeutic strategy.

TO THE EDITOR: A focus on defining genomic mutations at the whole population level in cancer may underestimate clonal plasticity in the tumor genome and how it generates genetic variation between individual single cells (SCs). This may have an impact on understanding cancer evolution and biological

Introduction: For chronic lymphocytic leukemia (CLL), especially in the lymph node (LN) setting where cells receive proliferative and pro-survival signals, in-depth studies of altered metabolism and its relationship with therapeutic responses are still lacking. Venetoclax, a BCL-2 inhibitor currently in wide clinical use for CLL, has shown high efficiency yet emerging resistance is a growing clinical problem. In cell line models, induced resistance to Venetoclax was accompanied by profound metabolic changes 1. This is in accordance with our earlier findings on metabolic and apoptotic changes that CLL cells undergo within the LN environment 2. In the current study, we performed RNA sequencing and applied fluxomics with 13C 6-glucose and 13C 5-glutamine to investigate in detail the metabolic routes in LN CLL. This led to studies to manipulate glutamine metabolism in a venetoclax resistance model. Methods: Peripheral blood (PB) samples from CLL patients were in vitro stimulated for 24 hrs by CD40 or B cell receptor (BCR), which are two potential key signals in LN. RNAseq analysis was compared with microarray data of paired PB/LN patient samples 3. For fluxomics, CLL cells were cultured for 2 hrs in medium containing either 5 mM 13C 6-glucose or 1 mM 13C 5-glutamine. Incorporation of 13C in metabolic intermediates was analyzed by LC-MS. For glutamine blockade, CLL cells were stimulated in presence of specific inhibitors of glutamine/glutamate metabolism or amino acid transporters. Cells were then treated with venetoclax, and viability was measured. Results: Gene expression profiles demonstrated that CLL cells obtained from LN tissue as well as after in vitro CD40 or BCR stimulation showed increased expression of gene sets involved in glycolysis, oxidative phosphorylation / citric acid cycle (OXPHOS/TCA) and amino acid metabolism as well as Myc activation. This confirmed that in vitro stimulation can be used to model the CLL LN setting. For unstimulated PB CLL cells, fluxomics data demonstrated low uptake of either glucose or glutamine, with 13C labelling close to zero for most metabolites. In contrast, both CD40 or BCR stimulation increased the uptake and utilization of glucose and glutamine. 13C labelling from glucose was detected in all glycolytic intermediates analyzed in both CD40- and BCR-stimulated CLL cells. Glucose was catalyzed to lactate and also partly converted to acetyl-CoA, which entered the TCA cycle. Additionally, labelling from glucose was also increased in several metabolites of the pentose phosphate pathway (PPP) suggesting it entered nucleotide synthetic routes. Compared to glucose, the contribution of glutamine was much higher in the TCA cycle in both BCR and CD40-stimulated cells. All intermediates of the TCA cycle were highly enriched with 13C from glutamine (Figure 1A). Combined, these data revealed that glutamine is the key metabolite to fuel the TCA cycle in LN CLL cells, and prompted us to study effects of glutamine blockade in conditions of Venetoclax resistance. It was found that venetoclax resistance induced by CD40 or BCR stimulation was clearly attenuated by glutamine uptake inhibition. CLL cells became re-sensitized to Venetoclax in both CD40- or BCR-stimulated samples, with an approximate 100-fold shift in IC50 (Figure 1B). Conclusions: Our study highlights the role of glutamine, in addition to glucose, in the metabolic reprogramming that CLL cells undergo in the LN (Figure 1C). These processes show potential for therapeutic targeting. Inhibition of glutamine import could contribute to dampen tumor microenvironment-induced Venetoclax resistance. References 1. Guièze, R. et al. Mitochondrial Reprogramming Underlies Resistance to BCL-2 Inhibition in Lymphoid Malignancies. Cancer Cell 36, (2019). 2. Chen, Z. et al. Effects of Ibrutinib on Metabolic Alterations and Micro-Environmental Signalling in Chronic Lymphocytic Leukaemia. Blood 136, (2020). 3. Herishanu, Y. et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-κB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 117, 563-574 (2011). Figure 1 Figure 1. Disclosures Forconi: AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria; Novartis: Honoraria; Gilead: Research Funding. van der Windt: Genmab: Current Employment. Kater: Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding; Abbvie: Honoraria, Other: Ad Board, Research Funding; Genmab, LAVA: Other: Ad Board, Steering Committee.

Purpose: Response rates to treatment of vulval intraepithelial neoplasia (VIN) with imiquimod and cidofovir are approximately 57% and 61%, respectively. Treatment is associated with significant side effects and, if ineffective, risk of malignant progression. Treatment response is not predicted by clinical factors. Identification of a biomarker that could predict response is an attractive prospect. This work investigated HPV DNA methylation as a potential predictive biomarker in this setting.Experimental Design: DNA from 167 cases of VIN 3 from the RT3 VIN clinical trial was assessed. HPV-positive cases were identified using Greiner PapilloCheck and HPV 16 type-specific PCR. HPV DNA methylation status was assessed in three viral regions: E2, L1/L2, and the promoter, using pyrosequencing.Results: Methylation of the HPV E2 region was associated with response to treatment. For cidofovir (n = 30), median E2 methylation was significantly higher in patients who responded (P ≤ 0.0001); E2 methylation >4% predicted response with 88.2% sensitivity and 84.6% specificity. For imiquimod (n = 33), median E2 methylation was lower in patients who responded to treatment (P = 0.03; not significant after Bonferroni correction); E2 methylation Conclusions: These data indicate that cidofovir and imiquimod may be effective in two biologically defined groups. HPV E2 DNA methylation demonstrated potential as a predictive biomarker for the treatment of VIN with cidofovir and may warrant investigation in a biomarker-guided clinical trial. Clin Cancer Res; 23(18); 5460–8. ©2017 AACR.

Chronic lymphocytic leukemia patients with mutated immunoglobulin heavy-chain genes (IGHV-M), particularly those lacking poor-risk genomic lesions, often respond well to chemoimmunotherapy (CIT). DNA methylation profiling can subdivide early-stage patients into naive B-cell–like CLL (n-CLL), memory B-cell–like CLL (m-CLL), and intermediate CLL (i-CLL), with differing times to first treatment and overall survival. However, whether DNA methylation can identify patients destined to respond favorably to CIT has not been ascertained. We classified treatment-naive patients (n 5 605) from 3 UK chemo and CIT clinical trials into the 3 epigenetic subgroups, using pyrosequencing and microarray analysis, and performed expansive survival analysis. The n-CLL, i-CLL, and m-CLL signatures were found in 80% (n 5 245/305), 17% (53/305), and 2% (7/305) of IGHV-unmutated (IGHV-U) cases, respectively, and in 9%, (19/216), 50% (108/216), and 41% (89/216) of IGHV-M cases, respectively. Multivariate Cox proportional analysis identified m-CLL as an independent prognostic factor for overall survival (hazard ratio [HR], 0.46; 95% confidence interval [CI], 0.24-0.87; P 5 .018) in CLL4, and for progression-free survival (HR, 0.25; 95% CI, 0.10-0.57; P 5 .002) in ARCTIC and ADMIRE patients. The analysis of epigenetic subgroups in patients entered into 3 first-line UK CLL trials identifies m-CLL as an independent marker of prolonged survival and may aid in the identification of patients destined to demonstrate prolonged survival after CIT., SCOPUS: ar.j, info:eu-repo/semantics/published

Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore disease-related/genetic features contributing to long-term outcomes. In 499 LRF CLL4 cases, a trial with >12 years follow-up, we employed targeted resequencing of 22 genes, identifying 623 mutations. After background mutation rate correction, 11/22 genes were recurrently mutated at frequencies between 3.6% (NFKBIE) and 24% (SF3B1). Mutations beyond Sanger resolution (KRAS mutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm TP53 mutations, we assessed the clinical impact of TP53 clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between TP53 mutations and either wild type or ≥12% VAF TP53mut cases. Secondly, we identified biallelic BIRC3 lesions (mutation and deletion) as an independent marker of inferior PFS and OS. Finally, we observed that mutated MAPK-ERK genes were independent markers of poor OS in multivariate survival analysis. In conclusion, our study supports using targeted resequencing of expanded gene panels to elucidate the prognostic impact of gene mutations.

In classic Hairy cell leukaemia (HCLc), a single case has thus far been interrogated by whole exome sequencing (WES) in a treatment naive patient, in which BRAF V(600) E was identified as an acquired somatic mutation and confirmed as occurring near-universally in this form of disease by conventional PCR-based cohort screens. It left open however the question whether other genome-wide mutations may also commonly occur at high frequency in presentation HCLc disease. To address this, we have carried out WES of 5 such typical HCLc cases, using highly purified splenic tumour cells paired with autologous T cells for germline. Apart from BRAF V(600) E, no other recurrent somatic mutation was identified in these HCLc exomes, thereby excluding additional acquired mutations as also prevalent at a near-universal frequency in this form of the disease. These data then place mutant BRAF at the centre of the neoplastic drive in HCLc. A comparison of our exome data with emerging genetic findings in HCL indicates that additional somatic mutations may however occur recurrently in smaller subsets of disease. As mutant BRAF alone is insufficient to drive malignant transformation in other histological cancers, it suggests that individual tumours utilise largely differing patterns of genetic somatic mutations to coalesce with BRAF V(600) E to drive pathogenesis of malignant HCLc disease.

Human multiple myeloma (MM) is characterized by accumulation of malignant terminally differentiated plasma cells (PCs) in the bone marrow (BM), raising the question when during maturation neoplastic transformation begins. Immunoglobulin IGHV genes carry imprints of clonal tumor history, delineating somatic hypermutation (SHM) events that generally occur in the germinal center (GC). Here, we examine MM-derived IGHV genes using massive parallel deep sequencing, comparing them with profiles in normal BM PCs. In 4/4 presentation IgG MM, monoclonal tumor-derived IGHV sequences revealed significant evidence for intraclonal variation (ICV) in mutation patterns. IGHV sequences of 2/2 normal PC IgG populations revealed dominant oligoclonal expansions, each expansion also displaying mutational ICV. Clonal expansions in MM and in normal BM PCs reveal common IGHV features. In such MM, the data fit a model of tumor origins in which neoplastic transformation is initiated in a GC B-cell committed to terminal differentiation but still targeted by on-going SHM. Strikingly, the data parallel IGHV clonal sequences in some monoclonal gammopathy of undetermined significance (MGUS) known to display on-going SHM imprints. Since MGUS generally precedes MM, these data suggest origins of MGUS and MM with IGHV gene mutational ICV from the same GC B-cell, arising via a distinctive pathway.

Introduction: Genomic-wide analysis of multiple myeloma (MM) reveals subclonal evolution as a major mechanism in disease evolution and progression, and interrogation of the entire functional genome at the single cell (SC) level is essential to fully map intra- clonal variation (ICV) and competition as the driver mechanism. To progress this, we have sought to establish whole exome sequencing (WES) in SCs in MM, initially in an index case of amp1q21 MM. Methods: CD138+ tumour cells and CD3+ T-cells were isolated from a presentation case of amp1q21 MM. Single MM and T cells were isolated for single cell WES. Whole genome amplification was performed using Qiagen REPLI-g Mini kit, and exome capture was performed using Agilent SureSelect. Libraries were 90 bp PE sequenced on an Illumina HiSeq2000 (BGI, China). Data was produced for bulk (1000 cells) MM and T cells, 20 MM SCs and 5 T cell SCs. Fastq were aligned to hg19 ref sequence using NovoAlignMPI (v3.02.03). Variant calling was performed using VarScan (v2.3.6) and variants were annotated using ANNOVAR. High confidence variants were called in the bulk tumour WES by pairwise comparison with bulk germline WES and by annotation against variant databases to exclude germline variants. Multiple quality control measures were employed to minimise false positives. Results and Discussion: SC WES generated raw data that were similar to bulk WES, with comparable mapping to target (69-76% SC vs. 70% bulk) and mean fold coverage (56.8-59.1x vs. 59.7x bulk). On average, 82% of the exome was covered sufficiently for somatic variant (SV) calling (! 5x), matching seminal published SC WES studies (70-80%) (Hou et al., Cell, 2012; Xu et al., Cell, 2012). We called 33 high confidence potentially deleterious SVs in the bulk tumour, 21 of which were also called in ! 1 SC exomes. SVs include mutations in genes involved in plasma cell differentiation, the MAPK pathway and known pathways in origins of B cellmalignancy. Random SVs were validated by Sanger sequencing with 100% concordance in the bulk (15/15) and SCs (55/55). ICV was apparent from the SC exome variant data. Total variant counts varied considerably among SCs and most variant positions had at least several cells where no evidence of the variant existed. Bulk WES lacks crucial information: We called 23 variants in ! 2 SCs that were absent in the bulk. Of these, 7/7 amplifiable variants were re-sequenced to obtain 100% concordance. These variants are of high interest as they reveal a marked occurrence of subclonal mu- tations in the MM tumour that are not identified by bulk WES. They indicate that the mutational status of the MM genome may be substantially underestimated by bulk WES. Conclusion: We establish the feasibility of SC WES as a method for defining true intraclonal genetic variation as a driver of cancer plasticity in MM.

Introduction Multiple myeloma (MM) is a largely incurable plasma cell malignancy characterised by marked genomic heterogeneity, in which chromosome 1q21 amplification (amp1q21) associates with poor prognosis. Genomic analysis using next generation sequencing has identified recurrent mutations, but no universal acquired somatic mutation(s) have emerged in MM, suggesting that understanding pathways of survival will require analysis of individual tumours in distinct disease subsets. To compound complexity of the problem, intraclonal variation (ICV), known as a major driver mechanism in cancer plasticity, in which clonal competitor cells undergo selection during disease evolution and progression by Darwinian principles, will need to be fully mapped at the genome level. Identifying the true level of ICV in a tumour will thus require analysis at the level of whole exome sequencing (WES) in single cells (SCs). In this study, we sought to establish WES methodology able to identify ICV in SCs in an index case of amp1q21 MM. Methods Cell selection and sequencing CD138+ tumour cells and CD3+ T-cells were isolated from a presentation case of amp1q21 MM as bulk populations to high purity (>97%). Single MM cells and normal T cells were individually isolated and used for single cell (SC) whole exome sequencing (WES). Whole genome amplification (WGA) was performed by multiple displacement amplification (Qiagen REPLI-g Mini kit), and exome capture was performed using Agilent SureSelect. Libraries were then 90 bp paired end sequenced on an Illumina HiSeq2000 (BGI, China). Data analysis Data was produced for bulk (1000 cells) MM and bulk germline T cells, twenty MM SCs and five T cell SCs. Raw data was aligned to hg19 reference sequence using NovoAlignMPI (v3.02.03). Variant calling was performed using SAMtools (v1.2.1) and VarScan (v2.3.6) and variants were annotated using ANNOVAR. High confidence variants were called in the bulk tumour WES by pairwise comparison with bulk germline WES. Variant lists were also cross-searched against various variant databases (CG46, 1000 genomes, dbSNP, esp650 and in-house database) in order to exclude variants that occur in the general population. Multiple quality control measures were employed to reduce the number of false positive calls. Results and Discussion Data and bioinformatics pipelines are of a high quality SC WES generated raw data reads that were similar to bulk WES of 1000 cells, with comparable mapping to Agilent SureSelect target exome (69-76% SC vs. 70% bulk) and mean fold coverage (56.8-59.1x vs. 59.7x bulk). On average, 82% of the exome was covered sufficiently for somatic variant (SV) calling (often considered as ≥ 5x), which was higher than seminal published SC WES studies (70-80%) (Hou et al., Cell, 2012; Xu et al., Cell, 2012). We identified 33 potentially deleterious SVs in the bulk tumour exome with high confidence bioinformatics, 21 of which were also identified in one or more SC exomes. The variants identified include suspected deleterious mutations in genes involved in MAPK pathway, plasma cell differentiation, and those with known roles in B cell malignancies. To confirm SV calls, randomly selected variants were validated by conventional Sanger sequencing, and of 15/15 variants in the bulk WES and of 55/55 variants in SCs, to obtain 100% concordance. Intraclonal variation in MM Significantly, ICV was apparent from the SC exome variant data. Total variant counts varied considerably among SCs and most variant positions had at least several cells where no evidence of the variant existed. Bulk WES lacks crucial information We identified an additional 23 variants that were present in 2+ SC exomes, but absent in the bulk MM tumour exomes. Of these, 30% (7 variants) were examined for validation, and were amplifiable in at least one cell to deliver 100% concordance with variant calls. These variants are of significant interest as they reveal a marked occurrence of subclonal mutations in the MM tumour population that are not identified by bulk exome sequencing. They indicate that the mutational status of the MM genome may be substantially underestimated by analysis at the bulk tumour population level. Conclusion In this work we establish the feasibility of SC WES as a method for defining intraclonal genetic variation in MM. Disclosures No relevant conflicts of interest to declare.

International audience; Vulval intraepithelial neoplasia is a precursor of vulval carcinoma, and is frequently associated with Human Papillomavirus (HPV) infection. Estimates of HPV prevalence in vulval intraepithelial neoplasia vary widely in the UK. The objective of this study was to assess HPV infection in a sample of women with vulval intraepithelial neoplasia, confirmed histologically, and determine the proportion of disease associated with HPV types targeted by prophylactic HPV vaccines. HPV infection was assessed in biopsies from 59 patients using the Greiner Bio-One PapilloCheck® DNA chip assay. Valid results were obtained for 54 cases. HPV infection was present in 43 of the 54 cases (79.6%: 95% CI 67.1-88.2%). The most common HPV types were HPV 16 (33/54: 61.1%), HPV 33 (8/54: 14.8%), HPV 6 (5/54: 9.3 %) and HPV 42 (3/54: 5.6%). The mean age of HPV positive women was significantly less than the mean age of HPV negative women. This is the largest UK series of vulval intraepithelial neoplasia in which HPV type has been investigated, and 34/54 (63.0 %, 95% CI: 49.6-78.6 %) cases were associated with HPV 16/18, which are targeted by current prophylactic HPV vaccines.