Studies on human papillomavirus (HPV) and other markers in the development and prognosis of HPV associated cancer

Background and aims: Human papilloma virus (HPV) is a risk factor for anogenital and oropharyngeal cancer (OPSCC) and commonly transmitted sexually although most infections are cleared without adverse effects. Notably, the past decades the incidences of HPV positive (HPV+), but not HPV negative (HPV-) tonsillar and base of tongue cancer (TSCC and BOTSCC), the two major OPSCC subtypes have both increased. For this reason, we wanted to follow HPV-prevalence. Before HPV vaccination a high prevalence of HPV was shown in the cervix and oral cavity of youth aged 15-23 years at a youth clinic in Stockholm 2008-2010, but later, with rising vaccine coverage, a decrease of HPV vaccine types was observed between 2013-2015. In parallel, in the mid-2010s many studies showed a link between vaginal microbiota and obstetric outcomes, inflammatory disease, as well as sexually transmitted disease. A few years later, several meta-studies and one DNA sequencing study abroad, showed an association between HPV prevalence and microbiota. In this context, of note, most HPV associated anogenital cancers go through three stages of development, pre-malignant stages, dysplasia, high-grade dysplasia/cancer in situ, to malignant stages, invasive cancer, and metastatic cancer. However, these stages are not well studied in HPV+ TSCC and BOTSCC and although there have been many biomarker studies in these tumours additional ones would be of use to better individualize treatment of these cancers. The aim of this thesis was therefore to follow up some of these findings. Approaches. In paper I, we followed up the HPV vaccination coverage and HPV prevalence at a youth clinic in Stockholm, to investigate vaccine effects. In paper II, we investigated possible effects of HPV status, age and vaccination status on the vaginal microbiota of women in a cohort from Uppsala and Stockholm. In paper III, we analysed and compared gene expression in high-grade dysplasia and invasive cancer in HPV+ and HPV- TSCCC/BOTSCC with particular emphasis on HPV status. In paper IV, we did whole-exome-sequencing on primary tumours of HPV+ TSCC/BOTSCC patients with and without recurrences, to identify similarities and differences between the groups as well as to identify markers of prognostic significance or candidates for targeted therapy. Results: In paper I, the proportion of HPV vaccinated women increased from 10.7% 2008-2010 to 82.1% 2017-2018. HPV-vaccine types were reduced overall and more in vaccinated than in unvaccinated women, but other high-risk HPV types still remained high. In paper II, microbial alpha-diversity was significantly higher for HPV+ compared to HPVpatients. Twice as many HPV+ than HPV- women had non-lactobacillus dominant vaginal microbiota compared to L. crispatus dominated vaginal microbiota and oncogenic HPVs were associated with non-lactobacillus dominant vaginal microbiota. In paper III, invasive and non-invasive tumours gene-expression were compared using immunohistochemistry (IHC) and RNA-panels. Forty genes showed differential expression, e.g. SPARC, psoriasin I, collagen-1 and galectin-1 and HPV+ and HPV- dysplasia was similarly differentiated from invasive cancer. In paper IV, a high-impact deletion on CDC27 was observed only in primaries of patients with relapse and 3 variants and 26 mutated genes, were present > 30% of all primaries regardless of prognosis. Conclusions. The presented studies in this thesis reaffirm the efficacy of the HPV vaccine programs in Stockholm, but with a remaining continuous prevalence of nonvaccine HR-HPV types. The results also suggest an influence from the HPV status on the vaginal microbiota make up. Furthermore, the data suggest that that HPV+ and HPVTSCC/ BOTSCC although not identical also likely have similar dysplastic cancer stages. Finally, there are differences between the mutational profiles of HPV+ TSCC/BOTSCC that re-occur compared to those that do not re-occur, but also here there are genes that are similarly altered in primary tumours of patients that are cured or that relapse

On the neuronal basis of cognition : cell-type specific circuitry and functions of the prefrontal cortex

This thesis recapitulates the history of research, and current knowledge, of the prefrontal cortex (PFC) in order to provide a context for the included scientific articles. The evident, but ill-defined, symptoms of a perturbation of the PFC is still a conundrum to neuroscientists. Though considered a source of cognition, or intellect, the quest to define an overall framework of how cognition is processed, or built, in the PFC, is very much an ongoing endower. The work presented in this thesis address both the structural architecture, as well as the electrophysiological properties, underlying the unique functions of the PFC. Chapter 2 of this thesis discusses the unique connectivity of the PFC and its relevance to a functional understanding of the neuronal computations present in the PFC. In this context, PAPER I reports the local and whole-brain connectivity scheme of discrete neuronal types within the PFC by the use of a novel rabies virus tracing system. Through carefully mapping monosynaptic inputs to four separate neuronal types, we describe that all connectivity traits defining the PFC, hold true for multiple neuronal types: the appearance of subnetworks within the PFC, the distinct thalamic innervation, and the high interconnectivity between PFC subregions. The third Chapter describes various electrophysiological properties present in the PFC, and more specifically, the occurrence of gamma oscillations, and their specific relevance to cognition. PAPER II and III report on the relevance of parvalbumin expressing interneurons for the generation of gamma oscillations in the rodent cortex. PAPER III further describes the presence of gamma oscillations during correct allocation of attention, and the frequency dependent activity of parvalbumin expressing interneurons. The temporal organisation of parvalbumin expressing interneurons, and the functional activity of excitatory neurons are, at this stage, only observations. However, the activity of parvalbumin expressing neurons was shown to be vital for the attentive state, and consequently, crucial for the network activity of the PFC. In summary, the work of this thesis, portrays cell type specific activity, as well as local and long-range circuitry of the rodent PFC. Although the concept of cognition may differ in appearance in mice as compared to humans, there is a common acceptance that key elements in the structure and function of the brain have been conserved through evolution, allowing for translatability. Ultimately, by carefully disentangling and observing small pieces of the circuitry and neuronal computation at a time, we can begin to build a framework for the neuronal underpinnings of cognition

Prefrontal Parvalbumin Neurons in Control of Attention

SummaryWhile signatures of attention have been extensively studied in sensory systems, the neural sources and computations responsible for top-down control of attention are largely unknown. Using chronic recordings in mice, we found that fast-spiking parvalbumin (FS-PV) interneurons in medial prefrontal cortex (mPFC) uniformly show increased and sustained firing during goal-driven attentional processing, correlating to the level of attention. Elevated activity of FS-PV neurons on the timescale of seconds predicted successful execution of behavior. Successful allocation of attention was characterized by strong synchronization of FS-PV neurons, increased gamma oscillations, and phase locking of pyramidal firing. Phase-locked pyramidal neurons showed gamma-phase-dependent rate modulation during successful attentional processing. Optogenetic silencing of FS-PV neurons deteriorated attentional processing, while optogenetic synchronization of FS-PV neurons at gamma frequencies had pro-cognitive effects and improved goal-directed behavior. FS-PV neurons thus act as a functional unit coordinating the activity in the local mPFC circuit during goal-driven attentional processing

Ovary transplantation method resulting in high reproductive performance in mice

There are mutant and transgenic mouse strains which lack the ability to breed, but where the females have functional ovaries. Ovary transplantation is an important tool for maintaining and producing crosses with these non-breeding strains.We have evaluated an ovary transplantation method by transplanting ovaries from females belonging to a non-reproduetive BALB/cByJ mutant mouse strain, All transplanted mice, 10 BALB/c.C57BL/6By, produced offspring and 94% of the progeny originated from the transplanted ovaries. The mean litter size and the mating period needed for productive mating to occur were similar to what is observed for corresponding control mice

Early Events in Xenograft Development from the Human Embryonic Stem Cell Line HS181 - Resemblance with an Initial Multiple Epiblast Formation

Xenografting is widely used for assessing in vivo pluripotency of human stem cell populations. Here, we report on early to late events in the development of mature experimental teratoma from a well-characterized human embryonic stem cell (HESC) line, HS181. The results show an embryonic process, increasingly chaotic. Active proliferation of the stem cell derived cellular progeny was detected already at day 5, and characterized by the appearance of multiple sites of engraftment, with structures of single or pseudostratified columnar epithelium surrounding small cavities. The striking histological resemblance to developing embryonic ectoderm, and the formation of epiblast-like structures was supported by the expression of the markers OCT4, NANOG, SSEA-4 and KLF4, but a lack of REX1. The early neural marker NESTIN was uniformly expressed, while markers linked to gastrulation, such as BMP-4, NODAL or BRACHYURY were not detected. Thus, observations on day 5 indicated differentiation comparable to the most early transient cell populations in human post implantation development. Confirming and expanding on previous findings from HS181 xenografts, these early events were followed by an increasingly chaotic development, incorporated in the formation of a benign teratoma with complex embryonic components. In the mature HS181 teratomas not all types of organs/tissues were detected, indicating a restricted differentiation, and a lack of adequate spatial developmental cues during the further teratoma formation. Uniquely, a kinetic alignment of rare complex structures was made to human embryos at diagnosed gestation stages, showing minor kinetic deviations between HS181 teratoma and the human counterpart

Non-CG DNA methylation is a biomarker for assessing endodermal differentiation capacity in pluripotent stem cells.

Non-CG methylation is an unexplored epigenetic hallmark of pluripotent stem cells. Here we report that a reduction in non-CG methylation is associated with impaired differentiation capacity into endodermal lineages. Genome-wide analysis of 2,670 non-CG sites in a discovery cohort of 25 phenotyped human induced pluripotent stem cell (hiPSC) lines revealed unidirectional loss (Δβ=13%, P<7.4 × 10(-4)) of non-CG methylation that correctly identifies endodermal differentiation capacity in 23 out of 25 (92%) hiPSC lines. Translation into a simplified assay of only nine non-CG sites maintains predictive power in the discovery cohort (Δβ=23%, P<9.1 × 10(-6)) and correctly identifies endodermal differentiation capacity in nine out of ten pluripotent stem cell lines in an independent replication cohort consisting of hiPSCs reprogrammed from different cell types and different delivery systems, as well as human embryonic stem cell (hESC) lines. This finding infers non-CG methylation at these sites as a biomarker when assessing endodermal differentiation capacity as a readout.We thank Kerra Pearce (UCL Genomics) for array processing, and Tim Fell and Jonathan Best (CellCentric), Jason Wray (UCL) and Rosemary Drake (TAP Biosystems) for discussions. We also thank Minal Patel, Chris Kirton, Anja Kolb-Kokocinski, Willem H. Ouwehand, Richard Durbin and Fiona M. Watt on behalf of the Human Induced Pluripotent Stem Cell Initiative (HipSci) funded by grant WT098503 from the Wellcome Trust and the Medical Research Council, for sharing data and materials. This work was supported in part by a TSB/EPSRC grant (TS/H000933/1). The Vallier lab is supported by the Cambridge Hospitals National Institute for Health Research Biomedical Research Center and an ERC Starting Grant (Relieve IMDS). F.A.C.S. is funded by a PhD studentship from Fundação para a Ciência e a Tecnologia (SFRH/BD/69033/2010). The Ferguson-Smith lab is supported by grants from the MRC and Wellcome Trust, and EU-FP7 projects EPIGENESYS (257082) and BLUEPRINT (282510). The Beck lab is supported by the Wellcome Trust (084071), a Royal Society Wolfson Research Merit Award (WM100023), and EU-FP7 projects EPIGENESYS (257082) and BLUEPRINT (282510).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1045

Characterization of Stem-Like Cells in Mucoepidermoid Tracheal Paediatric Tumor

Stem cells contribute to regeneration of tissues and organs. Cells with stem cell-like properties have been identified in tumors from a variety of origins, but to our knowledge there are yet no reports on tumor-related stem cells in the human upper respiratory tract. In the present study, we show that a tracheal mucoepidermoid tumor biopsy obtained from a 6 year-old patient contained a subpopulation of cells with morphology, clonogenicity and surface markers that overlapped with bone marrow mesenchymal stromal cells (BM-MSCs). These cells, designated as MEi (mesenchymal stem cell-like mucoepidermoid tumor) cells, could be differentiated towards mesenchymal lineages both with and without induction, and formed spheroids in vitro. The MEi cells shared several multipotent characteristics with BM-MSCs. However, they displayed differences to BM-MSCs in growth kinectics and gene expression profiles relating to cancer pathways and tube development. Despite this, the MEi cells did not possess in vivo tumor-initiating capacity, as proven by the absence of growth in situ after localized injection in immunocompromised mice. Our results provide an initial characterization of benign tracheal cancer-derived niche cells. We believe that this report could be of importance to further understand tracheal cancer initiation and progression as well as therapeutic development

Changes in Cervical Human Papillomavirus (HPV) Prevalence at a Youth Clinic in Stockholm, Sweden, a Decade After the Introduction of the HPV Vaccine

Aim: This study aimed to follow the impact of human papillomavirus (HPV) catch-up and vaccination on the very high cervical HPV-prevalence in women at a youth clinic in central Stockholm during the period 2008–2018.Background: 2008–2010, cervical HPV-prevalence (69.5%) and HPV16 prevalence (34.7%) were high in non-vaccinated women at a youth clinic in Stockholm. 2013–2015, after the introduction of the quadrivalent-Gardasil® HPV-vaccine, HPV16 and HPV6 prevalence had decreased. Here, cervical HPV-prevalence was investigated 10 years after primary sampling.Material and Methods: 2017–2018, 178 cervical swabs, from women aged 15–23 years old, were tested for 27 HPV types by a bead-based multiplex method. HPV-prevalence data were then related to vaccination status and age and compared to HPV-prevalence in 615 samples from 2008 to 2010 and 338 samples from 2013 to 2015 from the same clinic, and to HPV types in 143 cervical cancer cases during 2003–2008 in Stockholm.Results: The proportion of vaccinated women increased from 10.7% (2008–2010) to 82.1% (2017–2018). The prevalence of all 27 HPVs, all high-risk HPVs (HR-HPVs) and the combined presence of the quadrivalent-Gardasil® types HPV16, 18, 6, and 11, was lower in vaccinated compared to unvaccinated women (67.4 vs. 93.3%, p = 0.0031, 60.1 vs. 86.7%, p = 0.0057 and 5.8 vs. 26.7%, p = 0.002, respectively). Furthermore, HPV16 prevalence in non-vaccinated women 2017–2018 was lower than that in 2008–2010 (16.7 and 34.7%, respectively, p = 0.0471) and similar trends were observed for HPV18 and 11. In both vaccinated and non-vaccinated women, the most common non-quadrivalent-Gardasil® vaccine HR-HPV types were HPV39, 51, 52, 56, and 59. Together they accounted for around 9.8% of cervical cancer cases in Stockholm during 2003–2008, and their prevalence tended to have increased during 2017–2018 compared to 2008–2010.Conclusion: Quadrivalent-Gardasil® vaccination has decreased HPV-vaccine type prevalence significantly. However, non-vaccine HR-HPV types remain high in potentially high-risk women at a youth clinic in Stockholm

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Background and Aim: In 2010 HPV vaccination was subsidized in Sweden and in 2012 a national vaccination program against HPV16, 18, 6 and 11 was launched for girls ages 10-12 years. In parallel was a catch-up vaccination program for young women. To investigate base line HPV cervical and oral prevalence in non-vaccinated youth two studies were performed at a youth clinic in Stockholm 2008-2011. This project initiated 2013 aimed to follow HPV prevalence in youth since the previous studies, in the same population.   Materials and Methods: 117 women, of which 73% were HPV catch-up vaccinated donated 93 cervical samples and 117 oral samples, and 54 unvaccinated men donated 54 oral samples and 47 urinary samples. All samples were tested for 27 HPV types with a PCR based system and the data was compared to that obtained in 2008-2011. The categorical Fishers exact test was used for statistical analysis due to HPV-positive samples being n&lt; 5 for certain types.  Results and Conclusion: HPV16 cervical prevalence was significantly lower in the HPV vaccinated women compared to unvaccinated women (7% and 27% respectively, p=0.033) in the 2013 group. For HPV18 and HPV6 there was a significantly lower prevalence in the 2013 vaccinated group compared to the 2008-2010 unvaccinated group (1.5% vs. 10% respectively, p=0.021 and 1.5% vs. 8% respectively, p=0.048). Overall oral HPV prevalence for both genders, was lower in the 2013 group compared to that of 2009-2011, (2.3% and 9.1% respectively, p=0.005). Male urinary prevalence was low (6%) and not efficient to follow changes in specific HPV types. The data indicate that HPV catch-up vaccination was gradually exhibiting an effect, with significant decrease of cervical HPV16 prevalence