The immunosuppressive microenvironment in PDAC prevents tumor control but strategies to restore anti-cancer immunology, by increasing CD8 T cell activity, have not been successful. Here we demonstrate how inducing localized physical damage using ionizing radiation (IR) unmasks the benefit of immunotherapy by increasing tissue-resident NK (trNK) cells that support CD8 T activity. Our data confirms that targeting mouse orthotopic PDAC tumors with IR together with CCR5 inhibition and PD1 blockade reduces E-cadherin positive tumor cells by recruiting a hypofunctional NKG2C -ve NK population that supports CD8 T cell involvement. We show an equivalent population in human PDAC cohorts that represents an adaptive-like immunomodulatory trNK-cell that similarly supports CD8 T cell levels in a cDC1-dependent manner. Importantly, a trNK signature associates with survival in PDAC and solid malignancies revealing a potential beneficial role for trNK in improving adaptive anti-tumor responses and supporting CCR5i/αPD1 and IR-induced damage as a novel therapeutic approach.

Rs2073498 is a prevalent (~10% population) single nucleotide polymorphism (SNP) that leads to A133S missense mutation in tumor suppressor and Hippo Signaling Pathway scaffold protein RASSF1A. Recent reports have illustrated the crucial involvement of Hippo pathway proteins in lymphocyte recruitment, differentiation, and function. Using a BL/6 mouse model engineered with alanine-to-serine point mutation, we showed that RASSF1A133S is associated with systematic defects in regulatory T cells (Tregs). When blood was phenotyped through a 17-color spectral flow cytometry panel, both RASSF1A133S/WT and RASSF1A133S/A133S carriers had a smaller circulatory CD25+FoxP3+ Treg pool than WT controls. Magnetically sorted splenic T cells were also stimulated with PMA/Ionomycin ex vivo, and fewer Tregs from A133S carriers were expressing anti-inflammatory cytokine IL-10. Preliminary results further demonstrated that RASSF1A133S/WT has fewer intratumor FoxP3+ Tregs in subcutaneously injected MC38 colon adenocarcinoma tumor models. In contrast, A133S did not have any significant effects on thymic T cell maturation states, splenic T cell naïve/memory phenotypes, or Treg survival fitness measured by Fas/FasL and BCL-2 expression. Ex vivo stimulation also demonstrated that A133S carriers retain similar IL-2 sensitivity and phospho-STAT5 response when compared to the controls, although RASSF1 downstream kinases MST1/2 have been reported to amplify IL-2-STAT5 signaling in Tregs (Shi et al., 2018). These results collectively suggest that RASSF1A133S may leads to Treg imbalance and predispose rs2073498 carriers to Treg mediated autoimmune risks. Citation Format: Haonan Xu, Keaton Jones, Cameron Lang, Simone Lanfredini, Sophie Hughes, Eric O'Neill. RASSF1A133Sresulting from the prevalent SNP rs2073498 is associated with Treg defects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2885.

The basis of immune evasion, a hallmark of cancer, can differ even when cancers arise from one cell type such as in the human skin keratinocyte carcinomas: basal and squamous cell carcinoma. Here we showed that the basal cell carcinoma tumor–initiating cell surface protein CD200, through ectodomain shedding, was responsible for the near absence of NK cells within the basal cell carcinoma tumor microenvironment. In situ, CD200 underwent ectodomain shedding by metalloproteinases MMP3 and MMP11, which released biologically active soluble CD200 into the basal cell carcinoma microenvironment. CD200 bound its cognate receptor on NK cells to suppress MAPK pathway signaling that in turn blocked indirect (IFN-γ release) and direct cell killing. In addition, reduced ERK phosphorylation relinquished negative regulation of PPARγ-regulated gene transcription and led to membrane accumulation of the Fas/FADD death receptor and its ligand, FasL, which resulted in activation-induced apoptosis. Blocking CD200 inhibition of MAPK or PPARγ signaling restored NK cell survival and tumor cell killing, with relevance to many cancer types. Our results thus uncover a paradigm for CD200 as a potentially novel and targetable NK cell–specific immune checkpoint, which is responsible for NK cell–associated poor outcomes in many cancers.

RASSF1A promoter methylation has been correlated with tumor dedifferentiation and aggressive oncogenic behavior. Nevertheless, the underlying mechanism of RASSF1A‐dependent tumor dedifferentiation remains elusive. Here, we show that RASSF1A directly uncouples the NOTCH‐HES1 axis, a key suppressor of differentiation. Interestingly, the crosstalk of RASSF1A with HES1 occurs independently from the signaling route connecting RASSF1A with the Hippo pathway. At the molecular level, we demonstrate that RASSF1A acts as a scaffold essential for the SUMO‐targeted E3 ligase SNURF/RNF4 to target HES1 for degradation. The reciprocal relationship between RASSF1A and HES1 is evident across a wide range of human tumors, highlighting the clinical significance of the identified pathway. We show that HES1 upregulation in a RASSF1A‐depleted environment renders cells non‐responsive to the downstream effects of γ‐secretase inhibitors (GSIs) which restrict signaling at the level of the NOTCH receptor. Taken together, we report a mechanism through which RASSF1A exerts autonomous regulation of the critical Notch effector HES1, thus classifying RASSF1A expression as an integral determinant of the clinical effectiveness of Notch inhibitors. The RASSF1A tumor suppressor uncouples the NOTCH‐HES1 axis by triggering SNURF/RNF4‐mediated HES1 ubiquitination. Loss of RASSF1A promotes cancer stemness via NOTCH‐independent HES1 stabilization and confers resistance to γ‐secretase inhibitors. The RASSF1A tumor suppressor uncouples the NOTCH‐HES1 axis by triggering SNURF/RNF4‐mediated HES1 ubiquitination. Loss of RASSF1A promotes cancer stemness via NOTCH‐independent HES1 stabilization and confers resistance to γ‐secretase inhibitors.

Background and Aims Pancreatic ductal adenocarcinoma (PDAC) is characterised by advanced disease stage at presentation, aggressive disease biology and resistance to therapy resulting in extremely poor five-year survival <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression but exactly why is unclear and hindered by analysis of clinical samples. Methods Genome-wide epigenetic mapping of DNA modifications 5-hydroxymethylcytosine (5mc) and 5-hydroxymethylcytosine (5hmc) using oxidative bisulphite sequencing (oxBS). Bioinformatics using iCluster and mutational profiling to identify overlap with transcriptional signatures in FFPE from resected patients and confirmation in vivo. Results We find that more aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci including GATA6 that promote differentiated classical-pancreatic subtypes. We show that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5mc-hydroxylase TET2. Furthermore, we find that SMAD4 directly supports TET2 levels in the pancreas and classical-pancreatic tumors and loss of SMAD4 expression is associated reduced 5hmc, GATA6 and squamous-like tumors. Importantly, enhancing TET2 stability using Metformin and VitaminC/ascorbic acid (AA) restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNT-dependence in vitro and in vivo. Conclusions We identify epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data shows that restoring epigenetic control increases biomarkers of classical-pancreatic tumors and raises the possibility that combination of Vitamin C and Metformin may prolong survival in patients with squamous-like pancreatic cancer.

740 Background: SCALOP was a multi-centre phase II RCT where 114 patients with LAPC were received 3 cycles of Gemcitabine and Capecitabine (GEMCAP) and those with stable/responding disease (n = 74) were randomised to Gem-RT or Cap-RT. The trial showed superiority of Cap-RT. Baseline blood samples of randomised patients were analysed for 35 circulating biomarkers. In vivo study was undertaken with candidate biomarker (CCL5) to test actionability. Methods: Patient bloods were tested using R&D multiplexed magnetic Luminex assays and IGF-1, TGF-b1 and b-NGF DuoSet ELISA. Orthotropic KrasG12D;P53R172H;PDXcre (KPC) tumors were implanted in Bl6-mice and treated with Gem, CCR5-inhibitor (CCR5i) maraviroc (MV), PD1 inhibitor (PD1i), PD1i+MV alone and in combination with MRI guided small animal Radiotherapy (RT). Immunophenotyping was performed by IHC and Aurora Cytek spectral flow cytometry. Results: Baseline biomarker data was available on 63/74 randomised patients. Of the 35 biomarkers tested, only CCL5 was found to be significantly associated with OS with a median OS of 18.5 (95% CI: 11.76-21.32) vs 11.3 (9.86-15.51) months (low vs high), and HR 1.37 (95% CI:1.04-3.65; p = 0.037) in the Cox multivariable model. Treatment of orthotopic KPC tumors revealed that combination of MV+PD1i+RT resulted in tumour growth inhibition and a switch of tumour macrophages from M2 to M1 accompanied by increase in infiltration of cytotoxic CD8+ Tcells and NK cells. Conclusions: Previous pre-clinical studies reported CCL5-CCR5 axis as a poor prognostic marker and a possible cause of immune-resistance in pancreatic cancer. Herein we have demonstrated in prospectively collected clinical trial blood samples that high circulating CCL5 is associated with poor prognosis in LAPC. CCR5 inhibitor in combination with RT+PD1i may overcome immune-resistance, and should be tested in clinical trials. Clinical trial information: 96169987 .

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent form of pancreatic cancers with poor survival outcomes, due to late diagnosis, its propensity to metastasize, and lack of therapeutic efficacy under current chemotherapeutic regimens. Despite recent advancements in the field of immunotherapy, results from the clinic have demonstrated lack of efficacy when either radiotherapy (RT) or immunotherapy is used as a monotherapy in PDAC. However, recent publications revealed a synergistic effect on RT-induced immune modulation and reduced immune suppression when administered concurrently. Moreover, these reports demonstrate immune evasion in PDAC is dependent on the CCL5/CCR5 axis to recruit immunosuppressive T-regulatory cells (Tregs) into the tumor microenvironment. Therefore, targeting the migration of Tregs through modulation of CCL5/CCR5 can potentially inhibit tumor growth in pancreatic cancer. Aim: This preclinical study evaluates the impact of fractionated MR-image guided radiotherapy (MR-IGRT) in combination CCR5 inhibitor and simultaneous inhibition of the immune checkpoint axis PD1/PD-L1 in PDAC. Methods: For the purpose of this study we generated a syngeneic orthotopic pancreatic mouse model. Tumor cells derived from the Lox-Stop-Lox (LSL)-KrasG12D; LSL-Trp53R172H; Pdx1-cre (KPC) mouse model are injected in the tail of the pancreas. We are able to monitor tumor growth using a respiratory motion desensitized T2-weighted MRI imager, allowing the generation of high-resolution and high-contrast MRI data. Taking advantage of in-house developed technology, the MR-IGRT was delivered using the Small Animal Radiation Research Platform (SARRP) in combination with MRI imaging to deliver MR-guided fractionated radiotherapy. Concurrently with the radiotherapy, CCR5 inhibitor (maraviroc) and PD1 inhibitor were administrated at specific time points. To investigate the immunologic microenvironment, we developed a 17-color flow cytometry (FC) panel to immune-phenotype cytotoxic T, T regulatory, NK, NK/T and B cells, M-MDSC, PMN-MDSC, M1 and M2 macrophages in the peripheral blood and tumor infiltrate. Results/Conclusions: Using the established orthotopic mouse model and immune monitoring, we are investigating the therapeutic benefit of immunomodulation of the CCL5/CCR5 axis in combination with PD1/PDL1 and radiotherapy. Citation Format: Sophie Hughes, Simone Lanfredini, Asmita Thappa, Somnath Mukherjee, Eric O’Neill. Assessment of CCR5/maraviroc immunotherapy in combination with PD1 and MR-guided radiotherapy for treatment of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B69.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent form of pancreatic cancer with poor survival outcomes. Results from the clinic have demonstrated the lack of efficacy when either radiotherapy (RT) or immunotherapy are used as a monotherapy. Recent publications revealed a synergistic effect on RT-induced immune modulation and reduced immune suppression when the immunotherapy was administrated concurrently with RT in mouse models. Other publications demonstrate that immune evasion in PDAC depends on the CCL5/CCR5 axis to recruit immunosuppressive T-regulatory cells (Tregs) into the tumor microenvironment. Therefore, targeting the migration of Tregs through modulation of CCL5/CCR5 axis can potentially inhibit tumor growth in pancreatic cancer. Aim: This preclinical study is evaluating the impact of fractionated MR-Image Guided Radiotherapy (MR-IGRT) in combination CCR5 inhibitor and simultaneous inhibition of the immune checkpoint axis PD1/PD-L1 on pancreatic cancer. Methods: For the purpose of this study we generated a syngeneic orthotopic pancreatic mouse model. Tumor cells derived from the Lox-Stop-Lox (LSL)-KrasG12D; LSL-Trp53R172H; Pdx1-cre (KPC) mouse model are injected in the tail of the pancreas. We are able to monitor tumor growth using a respiratory motion desensitized T2-weighted MRI imager, allowing the generation of high-resolution and high-contrast MRI data. Taking advantage of in-house developed technology, the MR-IGRT was delivered using the Small Animal Radiation Research Platform (SARRP) in combination with MRI imaging to deliver MR-guided fractionated radiotherapy. Concurrently with the radiotherapy, CCR5 inhibitor (maraviroc) and PD1 inhibitor were administered at specific time points. To investigate the immunologic microenvironment, we developed a 17-color flow cytometry (FC) panel to immune-phenotype cytotoxic T, T regulatory, NK, NK/T and B cells, M-MDSC, PMN-MDSC, M1 and M2 macrophages in the peripheral blood and tumor infiltrate. Results/Conclusions: Using the established orthotopic mouse model, our aim is to investigate how the combination of MRI-guided radiotherapy and immune therapies can modulate the tumor microenvironment and the immune response in pancreatic cancer. This project is part of an ongoing preclinical study, and preliminary results will be presented at the meeting. Citation Format: Simone Lanfredini, Sophie Hughes, Asmita Thapa, Fiona Bangs, Jennifer Morton, Danny Allen, Veerle Kersemans, Paul Kinchesh, Sean Smart, Amy Elliot, James Thompson, Mark Hill, Somnath Mukherjee, Eric O'Neill. Assessment of CCR5i/maraviroc immunotherapy in combination with PD1 and MR-guided radiotherapy for treatment of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B30.

Purpose: Multi-slice scanning in the abdomen and thorax of small animals is compromised by the effects of respiration unless imaging and respiration are synchronised. To avoid the signal modulations that result from respiration motion and a variable TR, blocks of fully relaxed slices are typically acquired during inter-breath periods, at the cost of scan efficiency. This paper reports a conceptually simple yet effective prospective gating acquisition mode for multi-slice scanning in free breathing small animals at any fixed TR of choice with reduced sensitivity to respiratory motion. Methods: Multi-slice scan modes have been implemented in which each slice has its own specific projection or phase encode loop index counter. When a breath is registered RF pulses continue to be applied but data are not acquired, and the corresponding counters remain fixed so that the data are acquired one TR later, providing it coincides with an inter-breath period. The approach is refined to reacquire the slice data that are acquired immediately before each breath is detected. Only the data with reduced motion artefact are used in image reconstruction. The efficacy of the method is demonstrated in the RARE scan mode which is well known to be particularly useful for tumour visualization. Results: Validation in mice with RARE demonstrates improved stability with respect to ungated scanning where signal averaging is often used to reduce artefacts. SNR enhancement maps demonstrate the improved efficiency of the proposed method that is equivalent to at least a 2.5 fold reduction in scan time with respect to ungated signal averaging. A steady-state magnetisation transfer contrast prepared gradient echo implementation is observed to highlight tumour structure. Supplementary simulations demonstrate that only small variations in respiration rate are required to enable efficient sampling with the proposed method. Conclusions: The proposed prospective gating acquisition scheme enables efficient multi-slice scanning in small animals at the optimum TR with reduced sensitivity to respiratory motion. The method is compatible with a wide range of complementary methods including non-Cartesian scan modes, partially parallel imaging, and compressed sensing. In particular, the proposed scheme reduces the need for continual close monitoring to effect operator intervention in response to respiratory rate changes, which is both difficult to maintain and precludes high throughput.

Abstract Background: mRNA datasets have defined two molecular subtypes of pancreatic ductal adenocarcinoma (PDAC), classical and squamous, with distinct clinical characteristics and raising the possibility of subtype specific therapies (Collisson et al., 2019). The aggressive squamous subtype is characterized by poorer patient survival and loss of endodermal differentiation markers GATA6 and PDX1, but neither PDAC subtypes nor metastasis can be explained by genetic mutations alone (Reiter et al., 2018). Conversely, widespread epigenetic reprogramming is associated with progression to more aggressive phenotypes (McDonald et al., 2017), suggesting that aggressive molecular subtype is likely to be epigenetically driven. The role of DNA 5’methylcytosine (5’mc) in PDAC has been previously unclear due to an inability to distinguish this mark from the reciprocal activation mark DNA 5’hydroxymethylcytosine (5’hmC), mediated by Ten-eleven-translocation (TET) enzymes. Moreover, 5’hmc is dynamically regulated during pancreatic differentiation, but a role during PDAC initiation or progression has not been previously addressed. Methods: PDAC patients who underwent surgical resection at the Churchill Hospital, Oxford (n=146) had FFPE tumor tissue mutation comprehensively profiled by illumina hotspot array (300X), mRNA microarray, and the first epigenetic separation of 5’mc and 5’hmc on FFPE via oxidative bisulphite sequencing (oxBS) and illumina EPIC arrays. iCluster was used to highlight epigenetic PDAC subtypes and compare to existing TCGA datasets. Orthotopic models were employed to test whether the Squamous subtype is defined by 5’hmc loss and if this can be converted to the classical subtype in vivo. Results: Compared to healthy tissue, PDAC tumors demonstrate loss of 5’hmC at genes critical for pancreatic development and associated with PDAC progression, including MAPK signaling and TP53 targets. 5’hmC was preferentially lost in aggressive molecular subtypes (squamous) compared to the classical subtypes and was associated with SMAD4 mutations and reduced TET2. Overexpression of TET2 in squamous tumor cells restored 5’hmc and the expression of classical associated genes (e.g., GATA6 and PDX1), suggesting that 5’hmC is a master epigenetic regulator of PDAC molecular subtypes. Notably, we find TET2 stability is regulated by glucose levels, implying the widespread hypoglycemia seen in PDAC patients may contribute to progression of aggressive subtypes through loss of 5’hmC. Concomitantly, as metformin and vitamin C independently enhance endogenous TET2 activity, we find that this combination acts synergistically on squamous tumors to increase 5’hmC and expression of a classical-subtype phenotype, suggesting subtype switching is achievable in vitro and in vivo. Conclusion: These results identify 5’hmC as a regulator of molecular subtype that can be targeted in vivo using well-tolerated drugs. This abstract is also being presented as Poster A39. Citation Format: Michael Eyres, Simone Lanfredini, Frances Willenbrock, Asmita Thapa, Andrew Blake, Adam Burns, Ahmad Sabbagh, Aswin Abraham, Timothy Maughan, Zahir Soonawalla, Anna Schuh, Somnath Mukhergee, Eric O'Neill. Loss of TET2 activity results in epigenetic instability and drives PDAC molecular subtypes [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR02.

The pancreas is a gland composed mainly by endocrine and exocrine cells, giving rise to three main tumour types. Pancreatic neuroendocrine tumour or PNET arise from the endocrine portion of the pancreas. On the contrary, pancreatic exocrine neoplasms include pancreatic ductal adenocarcinoma (PDAC) and acinar cell carcinoma. PDAC is the most common type of pancreatic cancer and one of the leading causes of cancer-related death. It has been shown that less than 3% of PDAC patients have an overall survival of up to 5 years in the U.K. This mainly arises since the majority of patients diagnosed with PDAC present with advanced unresectable disease, which is highly resistant to all forms of chemotherapy and radiotherapy. Activating mutations of an isoform of the RAS protein, KRAS, are found in almost all PDAC cases and occur during early stages of malignant transformation. KRAS mutations play a critical role as they are involved in both initiating and maintaining PDAC development. The interaction of RAS with GDP/GTP along with its recruitment to the membrane affects transduction of its activating signals to downstream effectors. In this review, we aim to summarise different mutations of RAS and their prevalence in pancreatic cancer along with other RAS-induced tumours. In addition, we briefly discuss the genetically engineered mouse models that have been developed to study KRAS-mutated adenocarcinomas in the pancreas. These provide an opportunity to also address the importance of targeting RAS for better treatment response in PDAC patients along with the challenges incurred herein.

Many malignancies that occur in high excess in kidney transplant recipients (KTRs) are due to viruses that thrive in the setting of immunosuppression. Keratinocyte carcinoma (KC), the most frequently occurring cancer type in KTR, has been associated with skin infection by human papillomavirus (HPV) from the beta genus. In this report, we extend our previous investigation aimed at identifying the presence of active β-HPV infection in skin tumors from KTRs through detection of viral protein expression. Using a combination of antibodies raised against the E4 and L1 proteins of the β-genotypes, we were able to visualize infection in five tumors [one keratoacanthoma (KA), three actinic keratoses (AKs), and one seborrheic keratoses (SKs)] that were all removed from two patients who had been both transplanted twice, had developed multiple KCs, and presented with a long history of immunosuppression (>30 years). These infected tissues displayed intraepidermal hyperplasia and increased expression of the ΔNp63 protein, which extended into the upper epithelial layers. In addition, using a xenograft model system in nude mice displaying a humanized stromal bed in the site of grafting, we successfully engrafted three AKs, two of which were derived from the aforementioned KTRs and displayed β-HPV infection in the original tumor. Of note, one AK-derived xenograft, along with its ensuing lymph node metastasis, was diagnosed as squamous cell carcinoma (SCC). In the latter, both β-HPV infection and ΔNp63 expression were no longer detectable. Although the overall success rate of engrafting was very low, the results of this study show for the first time that β-HPV+ and ΔNp63+ intraepidermal hyperplasia can indeed progress to an aggressive SCC able to metastasize. Consistent with a series of reports attributing a causative role of β-HPV at early stages of skin carcinogenesis through ΔNp63 induction and increased keratinocytes stemness, here we provide in vivo evidence that these events are also occurring in the affected skin of KTRs. Due to these β-HPV-driven molecular pathways, the nascent tumor cell is able to acquire a high enough number of carcinogenic insults that its proliferation and survival will eventually become independent of viral gene expression.

Field cancerisation was originally described as a basis for multiple head and neck squamous cell carcinoma (HNSCC) and is a pre-malignant phenomenon that is frequently attributable to oncogenic human papillomavirus (HPV) infection. Our work on β-HPV-induced cutaneous squamous cell carcinomas identified a novel Lrig1+ hair follicle junctional zone keratinocyte stem cell population as the basis for field cancerisation. Herein, we describe the ability for HPV to infect adult tissue stem cells in order to establish persistent infection and induce their proliferation and displacement resulting in field cancerisation. By review of the HPV literature, we reveal how this mechanism is conserved as the basis of field cancerisation across many tissues. New insights have identified the capacity for HPV early region genes to dysregulate adult tissue stem cell self-renewal pathways ensuring that the expanded population preserve its stem cell characteristics beyond the stem cell niche. HPV-infected cells acquire additional transforming mutations that can give rise to intraepithelial neoplasia (IEN), from environmental factors such as sunlight or tobacco induced mutations in skin and oral cavity, respectively. With establishment of IEN, HPV viral replication is sacrificed with loss of the episome, and the tissue is predisposed to multiple cancer stem cell-driven carcinomas.

beta-Human papillomaviruses (HPVs) cause near ubiquitous latent skin infection within long-lived hair follicle (HF) keratinocyte stem cells. In patients with epidermodysplasia verruciformis, beta-HPV viral replication is associated with skin keratosis and cutaneous squamous cell carcinoma. To determine the role of HF keratinocyte stem cells in beta-HPV-induced skin carcinogenesis, we utilized a transgenic mouse model in which the keratin 14 promoter drives expression of the entire HPV8 early region (HPV8tg). HPV8tg mice developed thicker skin in comparison with wild-type littermates consistent with a hyperproliferative epidermis. HF keratinocyte proliferation was evident within the Lrig1 + keratinocyte stem cell population (69 vs. 55%, P < 0.01, n = 7), and not in the CD34+, LGR5+, and LGR6+ keratinocyte stem cell populations. This was associated with a 2.8-fold expansion in Lrig1+ keratinocytes and 3.8-fold increased colony-forming efficiency. Consistent with this, we observed nuclear p63 expression throughout this population and the HF infundibulum and adjoining inter-follicular epidermis, associated with a switch from p63 transcriptional activation isoforms to Delta Np63 isoforms in HPV8tg skin. Epidermodysplasia verruciformis keratosis and in some cases actinic keratoses demonstrated similar histology associated with b-HPV reactivation and nuclear p63 expression within the HF infundibulum and perifollicular epidermis. These findings would suggest that b-HPV field cancerization arises from the HF junctional zone and predispose to squamous cell carcinoma.

The aim of this study was to determine whether detection of β-HPV gene products, as defined in epidermodysplasia verruciformis skin cancer, could also be observed in lesions from kidney transplant recipients alongside the viral DNA. A total of 111 samples, corresponding to 79 skin lesions abscised from 17 kidney transplant recipients, have been analyzed. The initial PCR analysis demonstrated that β-HPV-DNA was highly present in our tumor series (85%). Using a combination of antibodies raised against the E4 and L1 proteins of the β-genotypes, we were able to visualize productive infection in 4 out of 19 actinic keratoses, and in the pathological borders of 1 out of 14 squamous cell carcinomas and 1 out of 31 basal cell carcinomas. Increased expression of the cellular proliferation marker minichromosome maintenance protein 7 (MCM7), that extended into the upper epithelial layers, was a common feature of all the E4-positive areas, indicating that cells were driven into the cell cycle in areas of productive viral infections. Although the present study does not directly demonstrate a causal role of these viruses, the detection of E4 and L1 positivity in actinic keratosis and the adjacent pathological epithelium of skin cancer, clearly shows that β-HPV are actively replicating in the intraepidermal precursor lesions of kidney transplant recipients and can therefore cooperate with other carcinogenic agents, such as UVB, favoring skin cancer promotion.