1. |
- Ljungström, Viktor, 1986-
(författare)
-
Exploring next-generation sequencing in chronic lymphocytic leukemia
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Next-generation sequencing (NGS) techniques have led to major breakthroughs in the characterization of the chronic lymphocytic leukemia (CLL) genome with discovery of recurrent mutations of potential prognostic and/or predictive relevance. However, before NGS can be introduced into clinical practice, the precision of the techniques needs to be studied in better detail. Furthermore, much remains unknown about the genetic mechanisms leading to aggressive disease and resistance to treatment. Hence, in Paper I, the technical performance of a targeted deep sequencing panel including 9 genes was evaluated in 188 CLL patients. We were able to validate 143/155 (92%) selected mutations through Sanger sequencing and 77/82 mutations were concordant in a second targeted sequencing run, indicating that the technique can be introduced in clinical practice. In Paper II we screened 18 NF-κB pathway genes in 315 CLL patients through targeted deep sequencing which revealed a recurrent 4 base-pair deletion in the NFKBIE gene. Screening of NFKBIE in 377 additional cases identified the mutation in ~6% of all CLL patients. We demonstrate that the lesion lead to aberrant NF-κB signaling through impaired interaction with p65 and is associated with unfavorable clinical outcome. In Paper III we sought to delineate the genetic lesions that leads to relapse after fludarabine, cyclophosphamide, and rituximab treatment. Through whole-exome sequencing of pre-treatment and relapse samples from 41 cases we found evidence of frequent selection of subclones harboring driver mutations and subsequent clonal evolution following treatment. We also detected mutations in the ribosomal protein RPS15 in 8 cases (19.5%) and characterization of the mutations through functional assays point to impaired p53 regulation in cells with mutated RPS15. Paper IV aimed at characterizing 70 patients assigned to three major subsets (#1, #2, and #4) through whole-genome sequencing. Besides recurrent exonic driver mutations, we report non-coding regions significantly enriched for mutations in subset #1 and #2 that may facilitate future molecular studies. Collectively, this thesis supports the potential of targeted sequencing for mutational screening of CLL in clinical practice, provides novel insight into the pathobiology of aggressive CLL, and demonstrates the clinical outcome and cellular effects of NFKBIE and RPS15 mutations.
|
|
2. |
- Handin, Niklas
(författare)
-
Proteomics informed investigation of human hepatocytes and liver tissue
- 2021
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A successful drug needs to display beneficial absorption, distribution, metabolism, excretion and toxicity (ADME-Tox) profile. It is therefore important to investigate these properties during the drug discovery process. The liver is of particular interest in ADME-Tox studies, as it is highly metabolically active and oral administrated drugs needs to pass the liver before reaching the systemic circulation. However, a dose of a drug that is efficacious and safe for one individual may be inefficacious or toxic, because of inter-individual variability. Therefore, it is important to investigate the ADME-Tox properties in a sufficiently large population. Investigations on ADME-Tox is usually done in in vitro cell models. Therefore, a variety of models to simulate liver functions have been developed and ranging from subcellular microsomes to complex 3D organoid cultures. This thesis investigates variability of ADME proteins in human liver tissue and in liver cell models.First, mass spectrometry based targeted proteomics was used to quantify ADME relevant proteins from 149 human liver samples. The observed inter-individual protein variability could not solely be explained by genotype. Therefore, a single transporter protein, the bile and drug transporting protein, NTCP, was investigate in detail. Non-genetic factors, e.g. smoking and alcohol consumption, and epigenetic factors such as DNA methylation, were found to contribute to the observed inter-individual variability of NTCP. Next, hepatocytes (PHH) were isolated from 54 human livers tissues and after which the hepatocytes where cryopreserved. The variable attachment efficiency of cryopreserved hepatocytes where investigated and an apoptosis inhibition protocol for restoration of attachment properties was developed. This protocol was also successfully applied to 3D cultured PHH spheroids resulting in increased ability to form 3D spheroids. The effect of culture conditions on the quality of the 3D cultures was also investigated. 3D PHH spheroids were formed and maintained in different, commonly used culture media. The spheroids were characterized by a variety of functional assays including global proteomics. The proteome analysis showed that while no epithelial to mesenchymal transitions was observed, 3D cultures maintained in fasting glucose and insulin levels resembling the in vivo situation showed a more liver-like phenotype with a high expression of ADME proteins and functional cytochrome P450 metabolism. Transporter kinetics were also investigated in the 3D cultured PHH. Finally, we investigated if global proteomics data from 56 human liver tissues could be deconvoluted to give information about the liver composition. The cell type proportions generated by deconvolution where similar to literature values. Liver samples that displayed deviating cell composition were identified. The deviating liver compositions were in agreement with clinical markers of inflammation in the patient´s blood samples and with altered extracellular matrix protein composition, comparable to that found in liver steatosis. In conclusion, this thesis have investigated variability in ADME proteins in human liver and in in vitro cultures of human hepatocytes, characterized cofounding factors for in vitro cultured hepatocytes and further extended drug disposition studies in 3D cultured hepatocytes.
|
|
3. |
- Österlund, Emerik, 1995-
(författare)
-
Prognostic and Predictive Factors in Metastatic Colorectal Cancer
- 2024
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The outcome for metastatic colorectal cancer (mCRC) patients has improved substantially in recent decades. This has chiefly been observed in study populations, and predominantly in left-sided primary tumours, which is why we wanted to study if and how survival has improved in the background population. It has also been seen that certain molecular subtypes are more common in population-based materials, and, thus, we studied the prevalence and effects of different molecular alterations.Paper I is a national population-based material of all 19 566 Swedish patients with a diagnosis of mCRC 2007-2016, 55% were male and 70% had synchronous metastases. Median overall survival (OS) for all patients was 14.0 months. An improvement could be seen over time, also in stratified analyses. OS was influenced by presentation of metastases, age, primary tumour location, and sex. All except sex remained statistically significant in a multivariable analysis. Differences of about one month in median OS were seen between healthcare regions, but these diminished over time.Paper II included all 765 patients from the Uppsala Region with a mCRC diagnosis 2010-2020. Right colon primary tumours were seen in 38%, left colon in 27% and rectum in 34%. BRAF-V600E mutations (mt) and deficient mismatch repair (dMMR) had a poor OS and were more common in right colon primary tumours. Primary tumour location did not affect OS in subgroups according to mutations in RAS or BRAF, nor in a multivariable analysis. Molecular alterations seem to be more important than primary tumour location for prognosis.Paper III studied KRAS-G12Cmt in three population-based and one real-world material. KRAS-G12C was seen in 2-4% of all tested and in 4-8% of all KRASmt. No differences in patient characteristics were observed between KRAS-G12C and other KRASmt. No differences in OS were seen between KRAS-G12C and other KRASmt, neither for all patients, nor in different treatment groups.Paper IV studied atypical BRAFmt (aBRAFmt) in two population-based and one real-world cohort. aBRAFmt was seen in 1-4% of the adequately tested patients in the different cohorts. aBRAFmt patients were predominantly male, had dMMR less often, more rectal primary tumours, and less peritoneal metastases compared with BRAF-V600Emt. Serrated adenocarcinomas were seen in about half of the aBRAFmt. OS was significantly better for aBRAFmt than in BRAF-V600Emt, but worse than for RASmt and RAS&BRAFwt patients. Nine aBRAFmt received epidermal growth factor receptor inhibitors without responses.
|
|
4. |
- Hammarström, Klara, 1990-
(författare)
-
Staging and therapy response in rectal cancer
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Every year, around 2,200 individuals are diagnosed with rectal cancer in Sweden. As a result of better tumour staging using magnetic resonance imaging (MRI), pre-operative radiotherapy (with or without chemotherapy), and improved surgery, outcome has improved substantially during the past few decades. Today less than 5% of patients experience a local recurrence. Treatment response is highly variable, up to 30% of patients have a complete remission (CR) after pre-treatment while others do not benefit from the treatment. The aim of this thesis was to investigate factors associated with CR in rectal cancer as accurate response prediction already at the time of diagnosis could enable a personalized treatment approach. For this purpose, an unselected rectal cancer cohort of approximately 1,200 patients diagnosed between 2010-2018 was built. Paper I provides a description of tumour stages and other MRI characteristics required for the treatment decision in the rectal cancer cohort. In this unselected patient population, most tumours belonged to the risk groups with intermediate or high risk of recurrence and are thus recommended to pre-treatment.In Paper II, the proportions of patients recommended pre-treatment according to different guidelines were investigated to better understand the wide variability in treatment seen worldwide. This study concluded that between 38% and 77% of non-metastatic patients are presently recommended pre-operative treatment according to 15 international guidelines, when strictly applied to our non-selected rectal cancer cohort. To achieve a more personalized treatment approach and a stricter use of pre-treatment, predictive factors of tumour remission are needed. In Paper III an evaluation of the predictive capacity of all clinical and pathological factors used in the staging of rectal cancer prior to treatment decision was done. In Paper IV a combination of clinical and sequencing data was used in analyses to further assess associations with CR and which factors impact prognosis. Tumour size, stage, tumour marker CEA and treatment were predictive of CR. Moreover, genetic factors such as mutated SMAD4 and SYNE1 were associated with CR but further investigations are needed to determine clinical relevance. Mutated KRAS was an independent predictor of non-CR. BRAF V600E mutation increased the risk of recurrence.
|
|
5. |
- Mathot, Lucy, 1986-
(författare)
-
From Tissue to Mutations : Genetic Profiling of Colorectal Cancer
- 2014
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Comprehensive characterisation of the mutational landscapes of solid tumours is a multistep process involving the collection of suitable samples, the extraction of nucleic acids and the preparation of these materials for mutational analyses. In this thesis, I aimed to develop a streamlined process for the analysis of colorectal cancer (CRC) patient samples in order to identify novel mutations that hallmark the development of advanced disease.Papers I and II outline a technique for serial extraction of nucleic acids from frozen tissue that we developed and subsequently implemented on a robotic platform to enable high-throughput processing. The extracted nucleic acids were validated in downstream processes relevant for genetic analyses, including traditional Sanger and next generation sequencing techniques.In Paper III, we developed a genotyping method based on multiplex ligation-dependent genome amplification. The method was designed such that InDel polymorphisms of between 30 and 70 % prevalence in a European population were selected and amplified in a multiplex PCR assay. DNA from 24 patient-matched colorectal tumour and normal tissues was genotyped and paired with a high match probability.In Paper IV, we performed targeted resequencing of 107 primary CRCs, of which approximately half developed metastatic disease or had distant metastases at the time of diagnosis. We chose to analyse 676 genes based on their involvement in key signalling pathways in CRC. We found an enrichment of mutations in the Eph receptor tyrosine kinase gene family in metastatic patients, indicating a potential role for these genes in CRC metastasis.This thesis outlines a series of procedures that can be employed in a high-throughput setting for the analysis of solid tumours. We applied these methods to the analysis of colorectal tumours and propose a link between novel somatic mutations and metastatic disease.
|
|
6. |
- Nunes, Luís, 1995-
(författare)
-
Prognostic and Predictive Somatic Mutations in Colorectal Cancer
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Colorectal cancer (CRC) is the third most incident and the second deadliest cancer worldwide. Even though CRC incidence is strongly correlated with age, it has been increasing in developing countries and younger individuals. Patients diagnosed with early stage local disease have 90% survival chance, but those detected with late stage metastatic disease have their survival odds reduced to 12-14%. Besides clinical and pathological tumour stages, molecular markers provide valuable information on patient outcome and help guide decision for different therapies. The aim of this thesis was therefore to identify and study somatic mutations and other genetic alterations in CRC samples to be used as prognostic and predictive biomarkers.In Paper I, we explored the mutational prevalence of known cancer genes in an unselected population cohort of metastatic CRC (mCRC) and compared this to what is reported in clinic-trial populations. This study demonstrated that BRAF-V600E, microsatellite instability-high (MSI-H) and right-sided tumour location were more common in populations than in previous clinical-based studies. Half of the patients had a tumour that was treatable with an FDA-approved targeted drug for mCRC, underlining the importance of evaluating targeted therapies upfront.In Paper II, we investigated what clinical and genetic factors lead to complete response (CR) after radiotherapy, alone or with chemotherapy, in a large population-based rectal cancer cohort, as well which factors impact overall survival and time to recurrence. Tumour stage, size, treatment, and mutation in SMAD4 or SYNE1 were predictors of CR while mutation in KRAS was a predictor of non-CR. BRAF V600E mutation increased the risk of recurrence.In Paper III, we studied the impact of somatic alterations in CRC through whole-genome and transcriptome sequencing. Successful sequencing was possible for 1,063 CRC primary tumours. Sequencing data analyses defined the somatic genomic and expression landscape and identified prognostic somatic coding and non-coding mutations, mutational signatures, structural variants and new expression subgroups. We could also link tumour hypoxia to different genetic alterations and subdivide the MSI samples in two distinct classes.In Paper IV, we integrated pan-cancer and pan-Ephrin mutational data to identify recurrent EPHB1 somatic mutations for further functional evaluation. Selected mutants and wild-type EPHB1 were transduced in DLD1 CRC cells and studied in compartmentalisation and phosphorylation assays. From the selected mutants, 7 lacked impact, 2 enhanced, and 6 reduced or strongly compromised cell compartmentalisation. This is, to date, the first integrative study of pan-cancer EPH receptor mutations followed by in vitro validation.
|
|
7. |
- Rendo, Verónica
(författare)
-
Targeting allelic loss in colorectal cancer
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Targeted cancer therapy exploits molecular differences between tumor and normal cells to selectively kill cancer cells. Whereas targeting of activated oncogenes has proved clinically useful, few current therapies exploit loss-of-function mutations in tumor suppressor genes or in the genome at large. This thesis explores the consequences of allelic loss affecting tumor suppressor genes and passenger genes in colorectal cancer (CRC), aiming to identify vulnerabilities that can be exploited for therapy. In Paper I we used genome editing to model inactivation of PRDM2 and showed that PRDM2 loss impacts cell growth and invasiveness, potentially mediated by genes involved in epithelial-to-mesenchymal transition. We confirmed the role of PRDM2 as a tumor suppressor gene in CRC and proved that c.4467delA inactivating mutations constitute a driver event in CRC.In Paper II we investigated whether the reduced allelic diversity resulting from loss of heterozygosity (LOH) in cancers could be exploited for therapy. We identified target genes by mapping prevalent alleles frequently lost in cancer and investigated NAT2 loss in CRC. Drug discovery efforts identified a compound selectively toxic to tumor cells with reduced NAT2 activity, providing proof of concept for LOH targeting by small molecule drugs.In Paper III, we aimed to widen the cohort of CRC patients eligible for NAT2 allele-selective chemotherapy. We determined NAT2 slow acetylator frequencies and LOH events in two independent cohorts by next-generation sequencing and genomic arrays. Next, we demonstrated enhanced response to allele-selective chemotherapy of tumor cells encoding additional prevalent NAT2 slow acetylator alleles, and developed a method for detection of NAT2 allelic loss suitable for clinical use.In Paper IV, we extended the search of therapeutic target genes by mining loss-of-function (LoF) alleles retained in tumors after LOH. This effort identified a prevalent splice site disruption in CYP2D6 as a putative target and motivated the development of cell model systems to identify compounds targeting CYP2D6 loss in cancer cells.In Paper V we characterized a set of 56 microsatellite stable CRCs by whole-genome sequencing in an attempt to understand the genetic causes leading to genomic instability and colorectal tumorigenesis. We confirmed the mutation frequencies of known CRC genes and identified for the first time the contribution of an unknown mutational process in 10% of the analyzed tumors.
|
|
8. |
- Ali, Muhammad Akhtar
(författare)
-
Understanding Cancer Mutations by Genome Editing
- 2014
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mutational analyses of cancer genomes have identified novel candidate cancer genes with hitherto unknown function in cancer. To enable phenotyping of mutations in such genes, we have developed a scalable technology for gene knock-in and knock-out in human somatic cells based on recombination-mediated construct generation and a computational tool to design gene targeting constructs. Using this technology, we have generated somatic cell knock-outs of the putative cancer genes ZBED6 and DIP2C in human colorectal cancer cells. In ZBED6-/- cells complete loss of functional ZBED6 was validated and loss of ZBED6 induced the expression of IGF2. Whole transcriptome and ChIP-seq analyses revealed relative enrichment of ZBED6 binding sites at upregulated genes as compared to downregulated genes. The functional annotation of differentially expressed genes revealed enrichment of genes related to cell cycle and cell proliferation and the transcriptional modulator ZBED6 affected the cell growth and cell cycle of human colorectal cancer cells. In DIP2C-/-cells, transcriptome sequencing revealed 780 differentially expressed genes as compared to their parental cells including the tumour suppressor gene CDKN2A. The DIP2C regulated genes belonged to several cancer related processes such as angiogenesis, cell structure and motility. The DIP2C-/-cells were enlarged and grew slower than their parental cells. To be able to directly compare the phenotypes of mutant KRAS and BRAF in colorectal cancers, we have introduced a KRASG13D allele in RKO BRAFV600E/-/-/ cells. The expression of the mutant KRAS allele was confirmed and anchorage independent growth was restored in KRASG13D cells. The differentially expressed genes both in BRAF and KRAS mutant cells included ERBB, TGFB and histone modification pathways. Together, the isogenic model systems presented here can provide insights to known and novel cancer pathways and can be used for drug discovery.
|
|
9. |
- Jiao, Xiang
(författare)
-
Somatic Mutations in Breast Cancer Genomes : Discovery and Validation of Breast Cancer Genes
- 2012
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Breast cancer is the most common cancer in women worldwide. However, the genetic alterations that lead to breast cancer are not fully understood. This thesis aims to identify novel genes of potential mechanistic, diagnostic or therapeutic interest in breast cancers by mutational analysis and whole-genome sequencing.In paper I, sequencing of 36 previously identified candidate genes in 96 breast tumors with patient-matched normal DNA determined the somatic mutation prevalence of these candidate genes and identified additional mutations in Notch, NF-κB, PI3K, and Hedgehog pathways as well as in processes mediating DNA methylation, RNA processing and calcium signaling.In paper II, comparison of massively parallel mate-pair sequencing results of a human genome before and after phi29-mediated multiple displacement amplification (MDA) revealed that MDA introduces structural alteration artifacts, with an emphasis on false positive inversions, and impairs the sensitivity to detect true inversions. Therefore, MDA has limited value in sample preparation for whole-genome sequencing for structural alteration detection.In paper III, massively parallel paired-end sequencing identified gene rearrangements in 15 hormone receptor negative breast cancers. Forty validated rearrangements were predicted to directly affect 30 genes, involved in epigenetic regulation, cell mitosis, signalling transduction and glycolytic flux. RNA interference-based assays revealed the potential roles in cell growth of some affected genes, among which DDX10 was implicated to be involved in apoptosis.In paper IV, a method for statistical evaluation of putative translocations detected by massively parallel paired-end sequencing was proposed. In an application of this method to analyse translocations detected by cancer genome deep paired-end sequencing, 76 putative translocations were classified into four categories, with the majority likely to be caused by mismapping due to repetitive regions.Taken together, this thesis provides insights into genes and pathways mutated in sporadic breast cancer genomes, which broaden our understanding of the genetic basis of breast cancer and may ultimately facilitate the diagnosis and treatment of this disease.
|
|
10. |
- Osterman, Erik, 1991-
(författare)
-
Colon Cancer, Prognosis After Surgery : What Are the Risks of Recurrent Disease, and How Do We Find Those at Risk?
- 2021
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Colon cancer is the fourth most common cancer worldwide with approximately 1.2 million yearly cases. Developments in the standard of care have improved prognosis. In Paper I the recurrence risk was investigated in a national material consisting of 14,325 colon cancer patients. Three fourths of stage II patients have a risk of approximately 11%, indicating that adjuvant chemotherapy can marginally improve prognosis. In stage III, one fifth of the patients have such a low risk of recurrence that the addition of a second chemotherapeutic drug, oxaliplatin with its risk for late toxicity, may be questioned. In Paper II emerging risk factors were investigated in a thoroughly staged and described material of 416 colon cancer patients from one county. All emerging risk factors correlated with an increased recurrence risk. Adjusting for established risk factors, pN-substage and postoperative carcinoembryonic antigen (CEA) correlated independently with recurrence. Paper III investigated the completeness and correctness of recurrences in the Swedish Colorectal Cancer registry in 2,893 patients from two counties. In patients operated more than 5 years ago 2% of recurrences were not registered. In Paper IV a nomogram for clinicians was developed using registry data to aid the interpretation of the recurrence risk and discussion with patients about treatment choices. It was validated in Norwegian cancer registry data and performed better than other available nomograms.Future investigations of the cohort from paper II are planned with immunohistochemistry, tumour-normal tissue sequencing and biomarkers.In summary, recurrence rates have decreased since the early 2000s and a large proportion of patients can probably be spared some or all adjuvant treatment. Established risk factors describe a large part of the risk, but there is room for improvement. The biomarker CEA taken after surgery could aid in the selection of patients to receive adjuvant treatment and guide follow-up. Adding other biomarkers might further improve the prediction of recurrence risk, though larger, prospective patient materials are needed.
|
|