Browsing by study line "Cancer"

Structural variants comprise a large number of variations occurring in the human genome and are detected in many diseases including cancers. To a limited extent, whole exome sequencing (WES) is capable of detecting structural variations (SVs) using algorithms and tools utilizing local assembly, split-reads, discordant read-pairs and read depth methods. However, due to the significantly large size of SVs compared to the reads produced and the presence of repetitive regions in the genome, identification of SVs presents a major challenge. 10X Genomics has developed a technology that requires very low amounts of DNA and uses a linked-reads approach to produce long reads. Recently, linked-read technology has shown promising results in resolving complex SVs. In this thesis, we aimed to assess whether linked-read exome sequencing is able to infer more comprehensive information in SVs compared to WES in multiple myeloma (MM). The disease model was chosen based on the presence of high numbers of SVs in MM patient tumor cells. Here, we report that linked-read sequencing has led to the identification of a potential novel translocation t(1; 14) that significantly impacts the change in expression of genes and could potentially have impact on the prognosis and treatment of multiple myeloma patients. By Long Ranger analysis we detected t(1;14) in six out of eight samples. Further, to study whether the translocation differentially affects the expression levels of any genes, differential gene expression was performed between t(1;14) positive versus t(1;14) wild type samples. The analysis resulted in 107 differentially expressed genes where 4 upregulated and 103 downregulated genes were found in the translocation positive samples. Among the downregulated genes, we found S100A8 and S100A9 genes which are previously shown to be associated with chemoresistance to PAD (bortezomib, doxorubicin and dexamethasone) therapy. The related breakpoints of the event were identified by Manta tool (SV caller) using both linked-read and WES. Therefore, linked-read information does not appear necessary to detect this event. In this study, we found that linked-read sequencing has certain advantages over WES such as low input DNA, increased number and quality of calls and breakpoint information. However, linked-read sequencing technique is limited to the detection of certain SV types in addition to increased cost of sequencing. These two factors must be considered before choosing linked-read sequencing over WES. Somatic mutations and clinically relevant SV were detected equally efficiently by both techniques.

Pään ja kaulan alueen syövät ovat seitsemänneksi yleisin söpäryhmä. Vuosittain siihen sairastuu 660 000 ja menehtyy 325 000 henkilöä maailmanlaajuisesti. Sen merkittävimmät riskitekijät ovat ikä, tupakointi, alkoholin kuluttaminen sekä HPV-infektio. Sen esiintyvyys on merkittävässä nousussa HPV-infektioiden yleistyessä sekä väestön ikääntymisen myötä. Gerastania on olennainen tekijä potilaan hoidon ja ennusteen kannalta. Siitä huolimatta tätä aihealuetta ja sen yhteyttä pään ja kaulan alueen syöpiin on tutkittu niukasti, sen merkittävyydestä huolimatta. Tällä hetkellä potilaan hoidon- ja ennusteenarvio on yksittäisen tai muutaman kliinikon standardoimattoman arvion käsissä. Tässä narratiivisessa kirjallisuuskatsauksessa perehdytään gerasteniaan ja pään ja kaulan alueen syöpien väliseen yhteyteen kokoamalla PubMed-tietokannasta aihealuetta käsitteleviä artikkeleita hyödyntämällä avainsanoja ’Head and Neck Neoplasms’, ’Frailty’ sekä ’Complications’ sekä ’Therapeutics’. Avainsana ’Therapeutics’ viittaa siis hoitoon, joka pään ja kaulan alueen syövissä on pääosin käsittää kirurgiaa sekä kemosädehoitoa. Tutkimuskysymystä käsitteleviä löydettiin 297 kappaletta, joista 66 hyödynnettiin tutkielman kirjoittamiseen. Tutkielma osoittaa, että pään ja kaulan alueen syöpiin ja niiden hoitoon liittyy monenlaisia komplikaatioita, joilla on osoitetusti huono ennustevaikutus erityisesti gerastenian kanssa. Tutkimus myös osoittaa, että on toivottavaa arvioida systemaattisesti potilaiden gerasteniaa kvantitatiivisesti, sillä pään ja kaulan alueen syöpien potilaat ovat usein hauraita potilaita, joiden kohdalla gerastenian huomioimattomuus voi johtaa väärään tai kohtalokkaaseen hoitoon.

Epithelial Ovarian cancer remains to be one of the leading causes of cancer-related deaths in women, and 70-80% of EOC-related deaths are attributed to High-Grade Serous Ovarian Cancer (HGSC). The 5-year survival rate of HGSC remains concerning at 50%. Approximately 50% of HGSC patients exhibit impaired Homologous Recombination (HR) DNA repair, a high-fidelity double-stranded DNA damage repair mechanism. Patients with Homologous Recombination Deficient (HRD) tumors exhibit better responses to DNA-damaging platinum agents and Poly (ADP-ribose) polymerases (PARP) inhibitors. The success of these treatments is attributed to the inability of HRD tumors to repair DNA damage, ultimately leading to cell death. It is expected that Homologous Recombination Proficient (HRP) tumors have functional repair mechanisms to eliminate this damage. Therefore, they derive no durable benefit from DNA-damaging agents and PARP inhibitors, resulting in a poor prognosis. DNA damage can be identified by the presence of pHistoH2AX (phosphorylated H2AX protein). This protein is formed at an early stage in response to the presence of Double-Strand Breaks (DSBs), and its interaction with MDC1 is critical for the recruitment of subsequent DNA repair proteins. Interestingly, Immuno-Fluorescent Imaging has revealed the presence of pHistoH2AX in chemo naïve HRP tumors. This raises questions about the origin of this endogenous DNA damage in HRP tumors and whether it signifies underlying vulnerabilities that could be exploited for therapeutic purposes. To address these questions, my thesis aimed to identify the co-expression markers of pHistoH2AX. To achieve this objective, I analyzed existing data from Cytometry by Time of Flight and Cyclic Immunofluorescence experiments to identify interesting molecular candidates. These experiments gave rise to several interesting candidates including pSTAT3, PDL1, pAkt, pEGFR, Cyclin E1 and Vimentin as potential co-expressed markers in HRP tumors. I further validated the co-expression of pSTAT3 and pHistoH2AX in HGSC patient tissue sections by immunofluorescence staining. This study holds significant relevance for identifying alternative molecular targets that could potentially improve the survival outcomes of patients with HRP HGSC. Future studies, encompassing larger cohorts of HRP patients, may unveil suitable subpopulations that could benefit from targeting these identified proteins, paving the way for the development of more effective therapeutic strategies in the management of HRP HGSC.

Although approximately 80% of children with cancer survive five years after diagnosis, many childhood cancer survivors suffer from side effects and long-term health complications caused by the treatments. In addition, the diseases can often recur due to ineffective therapy. In pediatric sarcoma treatment, the recurrence of the disease is one of the major challenges. In rhabdomyosarcoma (RMS) 30% of cases end up relapsing and the cure rate is low with current therapies. Functional precision medicine approach is of interest especially with children since childhood cancers are likely diseases of upregulation or downregulation of protein expression with an overall low mutational burden and a lack of identifiable targets. In functional precision medicine the information from drug sensitivity and resistance testing (DSRT) of patient-derived cells (PDCs) is combined with other molecular tumor data, such as genomic and transcriptomic data, to better guide the selection of personalized treatments. In this study, we first established the PDC culture conditions for two longitudinal tumor samples of a young patient with RMS. DSRT was performed to age-matched healthy control fibroblasts and aortic smooth muscle cells, four RMS cell lines with different molecular backgrounds, and as a proof-of-concept study, to two longitudinal tumor samples of the RMS patient, to find compounds able to inhibit RMS cell viability. In addition, the clinically relevant genetic findings of the patient were highlighted. Specific drug responses relating to the molecular characteristics of the RMS cell lines were observed, such as MEK inhibitors in RAS mutated fusion-negative RMS cells. The findings of this work show that the DSRT assay can be utilized to discover RMS specific drug vulnerabilities, and it reveals the importance of proper control cells when using DSRT. This study provides evidence that combination of DSRT and NGS-based diagnostics may provide additional value for cancer treatment.

Transposable elements (TEs) are repetitive DNA elements that have an autonomous capability of replicating and inserting themselves into new loci within genomes. TEs have often been thought to be a part of the non-functional “junk DNA”, but advances in next-generation sequencing technology has revealed that TEs have rich biochemical functions: Specific TEs bind a large fraction of transcription factors and harbor marks of active chromatin in human genomes. However, there have been few genome-wide functional enhancer activity studies on the role of TEs in gene regulation, especially in the context of human cancers. In normal cellular homeostasis, TE activity is tightly controlled by epigenetic mechanisms. In contrast, the cell state in cancer genomes is often permissive for TE activation: genome instability and mutations, notably p53 inactivation, and nonmutational epigenetic reprogramming such as DNA hypomethylation are common characteristics of cancers. TE transcription, somatic retrotransposition and activation of cryptic promoter elements occur frequently in tumors. TE activation is highly heterogeneous between cancer types: for example gastrointestinal tract cancers such as colorectal cancer show high somatic insertion activity, whereas retrotransposition is rare in hematolymphoid malignancies. Due to the widespread TE activation in cancers, we posited that this may also be seen in the activity of cryptic TE enhancers, with specific active families of TEs characteristic to different cell types due to lineage-specific TF binding. We asked if TEs contribute to the enhancer landscape of cancers and to which extent, what are the differences between cancers in the activity and transcription factor binding and whether TE enhancers may have a role in tumorigenesis and the regulation of cancer-specific genes. To functionally study TEs, we utilized a high-resolution, unbiased, genome-wide massively parallel reporter assay (MPRA). We utilized colorectal and hepatocellular cancer cell lines to study the differences and similarities between TE activation and combined the MPRA data with orthogonal epigenetic data to study the in vivo signatures of the TEs. We found that both cell lines show common and highly enriched TE subfamilies that were mostly specific for p53, as well as TEs that were highly unique in both cell lines. By using in vitro methylated MPRA libraries, we found that CpG methylation has relatively minor a role in regulating the enhancer activity of some TE subfamilies in the reporter assay. By comparing the epigenetic context of the TEs, we found that especially colorectal cancer has specific highly active TE subfamilies with signatures of canonical active enhancers. We also used an in silico model to predict TE enhancer to gene contacts and found that these subfamilies regulated genes that were frequently overexpressed. Thus, we present the widespread functional activity of TE enhancers in cancers, providing evidence for further functional validation of TEs and their effects on transcriptional programs and especially dysregulation of gene expression in cancer.

High-grade serous ovarian cancer (HGSC) presents a complex clinical challenge as it is often diagnosed at advanced stages. Neoadjuvant Chemotherapy (NACT) is commonly used to treat advanced stage (III and IV) patients who cannot undergo primary debulking surgery. However, 80% of them experience relapse and develop resistance to platinum-based chemo therapies. While NACT alters the tumor immune microenvironment in a treatment response specific manner, its underlying mechanisms remain unclear. Understanding the effect of NACT on tumor microenvironment (TME) is crucial to identify novel biomarkers and develop effective therapeutic strategies. Emerging spatially resolved methodologies, including highly multiplexed-imaging and spatial gene-expression profiling approaches provide novel information regarding spatial interactions and mechanisms operating at single-cell level. However, neither of these methods can offer a comprehensive view of the tumor immune landscape while also revealing the molecular mechanisms behind it. To overcome this limitation, this thesis proposes a novel method integrating tissue cyclic immunofluorescence (tCycIF) and GeoMx DSP spatial transcriptomics. tCycIF is a high-throughput multiplexed imaging method, while GeoMx DSP offers sequencing information at near to single-cell resolution in well-defined regions of interest (ROIs), enabling the dissection of the underlying molecular mechanisms. Here I set out to explore the effect of NACT on tumor stroma interface (TSI) and the surrounding tumor and immune cells, particularly IBA1+ macrophages, and CD8+ T cells. HGSC patient-derived pre- and post-NACT FFPE blocks were acquired from Helsinki University Biobank. To preserve morphological and spatial features, adjacent tissue sections underwent tCycIF and GeoMx experiments. Potential regions for spatial transcriptomics (Pre ROIs) were successfully delineated based on tCycIF staining patterns, enabling the identification of IBA1+ macrophage and CD8+ T cell rich areas within the TSI. By overlaying a crop of the tCycIF image onto the GeoMx scan, areas with distinct similarity degrees between tCycIF and GeoMx were generated. The GeoMx-ROIs were ultimately selected based on the Pre-ROIs and their similarity degree with tCycIF scanned images. Tumor and stroma segmentation was performed through a custom segmentation method guided by tumor specific marker pan-cytokeratin staining in GeoMx images, defining distinct spatial compartments for sequencing. Finally, the image-derived data from both techniques was integrated in a single file, enabling a combined subsequent analysis. The novel pipeline developed in this study opens promising research possibilities, as tCycIF-guided ROI selection allows for precise targeting of cell neighborhoods and structures after performing a thorough microenvironment exploration. This approach can potentially be adapted for other uses in a wide range of biomedical fields, beyond the focus of HGSC.

The current 5-year survival rate of OSCC patients is around 50%. There are no diagnostic tests or markers and the diagnosis is based on histopathologic samples only, which postpones the time of detection and worsens the prognosis of the patient greatly. New diagnostic methods and tools are required to improve the survival rate. This study aimed to find possible biomarkers and develop a new diagnostic method for OSCC by comparing the serum proteomic expression of tongue cancer patients and healthy controls with label-free liquid-chromatography mass spectrometry. The results showed small, but statistically significant difference in protein expression between the patients and healthy controls, as well as a clear separation between the groups based on the peptide data. In addition, no specific networks or cellular pathways were highlighted for OSCC compared to other types of cancers. These results didn’t introduce considerable advances into the diagnostics of OSCC but showed a possibility for finding further distinctive differences between the OSCC patients and healthy controls.

High-grade serous ovarian cancer (HGSC) is the most aggressive subtype of ovarian cancer. Most patients are diagnosed at an advanced stage, resulting in a poor prognosis. Although some targeted therapies have made good progress, immune checkpoint inhibitors (ICIs) still show limited efficacy in HGSC treatment. This is closely associated with the high heterogeneity and unique immunosuppressive tumor microenvironment (TME) of HGSC. As local immune centers in the TME, tertiary lymphoid structures (TLSs) contain diverse types of immune cells and play a crucial role in the anti-tumor immune process. Nonetheless, the spatial immune landscape of TLSs and key gene expression signatures affecting prognosis have not been fully elucidated. In recent years, spatially resolved gene expression profiling of clinical samples has provided insights into the transcriptome at high resolution. However, despite revealing a series of response mechanisms of anti-tumor immunity, these methods still have many limitations in non-single cell resolution, specific tissue sample types, and high cost. Here in this thesis, I established Nano-Pick, a novel method based on precise microtissue extraction and the nCounter analysis system. Nano-Pick enables cost-effective and rapid spatial gene expression profiling using formalin-fixed paraffin-embedded (FFPE) samples. All TLSs-enriched FFPE slides were obtained from patient-derived HGSC samples at Helsinki University Hospital (HUH). With the help of tissue cyclic immunofluorescence (t-CycIF), a highly multiplexed imaging method, selected regions of interests (ROIs) were annotated in different zones of TLSs. These ROIs were accurately transferred to adjacent tissue sections, guiding the target region selection for microtissue extraction. An improved immunofluorescence staining method was used to successfully label cancer cells, macrophages, and immune cells without antigen retrieval. With the optimized combination of parameters, efficient and consistent precise picking for different cell types was achieved. mRNA extracted from the collected microtissues was validated by qPCR for the range of pre-amplification cycles, confirming its suitability for nCounter gene panel analysis. Therefore, Nano-Pick offers the opportunity for higher-resolution spatial analysis and interactions of immune cell populations in the TME, especially in the TLSs. This low-cost, reliable and sensitive spatial gene expression profiling method has broad application prospects in many fields, with the potential to advance the development of precision medicine.

Background: Oral contraceptive (OC) use may increase the risk of specific cancers and mortality. The aims of this study were to investigate the association between OC use and its duration with the risk of breast cancer, to examine the overall mortality associated with OC use and its duration, and finally to identify sociodemographic characteristics of OC use. Methods: Data are derived from the Older Finnish Twin cohort consisted of monozygotic and same-sexed dizygotic twin pairs born before 1958. We included N = 9,607 Finnish twin women aged 18 – 49 years old with information on OC use and other covariates. The information on OC use, reproductive, and lifestyle factors was collected using a mailed questionnaire. The information on breast cancer incidence was obtained from the Finnish Cancer Registry and the data on mortality was collected from the national Population Information System and Statistics Finland. We used Cox proportional hazards regression to estimate the association between OC use and its duration with risk of breast cancer and overall mortality while controlling for potential confounders. Also, we used logistic regression to identify sociodemographic characteristics of OC use. All tests of statistical significance were two-sided. Results: A total of 758 women developed breast cancer during median follow-up of 42.6 years. Women who ever used OC had 20% greater risk of developing breast cancer than women who never used (HR =1.20, 95% CI = 1.02 to 1.40, P = 0.02). Women who used OC for more than 5 years had greater risk of developing the disease than those who used OC for less than 2 years (HR = 1.11, 95% CI = 0.85 to 1.46), however, the results did not reach the statistical significance. Mortality did not significantly different between women who had ever used OC with those who had not used OC while controlling for potential confounders. Current smokers and women who consumed alcohol more than 10 gram/day had the highest odds of ever using OC. Conclusion: Our results suggest that OC use slightly increases the risk of breast cancer, however, no evidence from this study indicates that OC use adversely affect long-term risk for mortality.

Kaposi sarcoma (KS) is a cancer characterized by spindle cells thought to originate from endothelial cells. The most aggressive form is AIDS-related KS, which is one of the most common cancers in Sub-Saharan African regions with high HIV burden. The causative agent of KS, Kaposi Sarcoma Herpesvirus (KSHV), displays a biphasic lifestyle of latent and lytic phases. During the latent program, the viral DNA is tethered to the host chromatin without integration, through the viral protein LANA. This mechanism allows the KSHV genome to exploit the human cellular replication machinery to replicate and maintain viral DNA genomes. Recent, unpublished findings suggest that this mechanism is supported by interactions between LANA and the host transcription factor SOX18. The Ojala lab has shown that this key developmental factor SOX18 is significantly upregulated in KSHV-infected cells. Importantly, SOX18 inhibition by a small molecule inhibitor SM4 decreases the intracellular viral DNA copies and characteristic infected spindle cell phenotype. However, the mechanism behind the upregulation of SOX18 is not understood. Additionally, a previous BioID screen for SOX18 protein interactions discovered that an important structural protein and the ATPase of the host chromatin remodeling cBAF complex, ARID1A and BRG1, respectively, were the highest confidence interaction partners in KSHV-infected cells. Therefore, it was hypothesized that SOX18 and the cBAF complex, and their interactions, co-operate in viral DNA replication and could represent promising therapeutic targets for KS and other SOX18-dependent diseases. This study found that host signaling pathway NF-κB may be responsible for SOX18 upregulation. A BrdU pulldown assay with ectopic SOX18 expression showed enhanced KSHV DNA synthesis, which could be hampered with SM4 inhibition. These results further elucidate the role of SOX18 in viral genome replication. Furthermore, proximity ligation assays and immunofluorescence were performed to investigate interactions between SOX18, KSHV LANA and cBAF in KSHV-infected cells. Cell viability assays, protein expression by Western blotting, and KSHV genome copy quantification upon treatment with cBAF inhibitors were performed. In addition to SM4, inhibition of cBAF led to a regression of the spindle cell phenotype and reduced KSHV genome copies and showed that KSHV-infected cells are sensitized to cBAF inhibition. Infection with a constitutively latent virus supported these results. This thesis sheds light on the mechanism of SOX18 interaction with LANA and host chromatin remodeler in viral latent DNA replication and provides a deeper insight into possible future therapeutic strategies for KS.