| 1. |
- Malmström, Annika, 1957-
(författare)
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Studies for Better Treatment of Patients with Glioma
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In Sweden annually over 500 people will be diagnosed with the malignant brain tumor glioma. They are graded from I-IV. The majority are glioblastoma (grade IV) (GBM), these being the most aggressive type. Median survival for those treated with standard of care is expected to be around 15 months. This tumor will mainly affect those 60 years or older.The studies in this thesis focus on treatment of patients with malignant gliomas grade III and IV. The aim of the studies is to improve the care of glioma patients. Papers I and II explored different therapeutic options in randomized trials, to facilitate individualized treatment recommendations. Findings from studies I and II, together with additional trials, demonstrated the importance of analyzing the tumor marker O6-methylguanine DNA methyltransferase (MGMT) methylation status for survival of GBM patients treated with Temozolomide (TMZ). The third paper investigated how the analysis of this marker is implemented internationally.The first study (paper I, Nordic trial) investigated treatment options for patients 60 years or older with GBM. The trial compared standard radiotherapy (SRT) over 6 weeks versus hypofractionated radiotherapy (HRT) over 2 weeks versus single agent TMZ administered in up to six 4 weekly cycles. In all, 342 patients were included in the trial. This study demonstrated that those randomized to TMZ had superior survival as compared to SRT. In addition, quality of life (QoL) data also suggested a better QoL for TMZ treatment than for radiotherapy. The benefit of TMZ treatment seemed to be limited to those with the tumor molecular marker MGMT methylated (inactivated).The second trial (paper II, Neoadjuvant trial) studied whether integrating TMZ treatment with SRT for patients younger than 60 years with GBM (grade IV) and astrocytoma grade III would confer a survival benefit, if administered postoperatively, before the start of SRT (neoadjuvant). TMZ was provided for 2-3 four weekly cycles followed by SRT to patients randomized to neoadjuvant treatment and was compared to postoperative SRT alone. Although this trial could not illustrate any advantage of delaying the start of SRT while administering TMZ for the study cohort in general, for those included as astrocytoma grade III the median survival was found to be superior by 5 years when randomized to neoadjuvant TMZ. This trial also confirmed the importance of MGMT promoter methylation for the efficacy of TMZ.The third study (paper III) investigated international practices for analyzing tumor MGMT promoter methylation status. MGMT analysis can be conducted by various laboratory methods, which in some cases can provide opposing results regarding the MGMT methylation status of the patient´s tumor. This can lead to incorrect treatment recommendations. To establish which methods and cut-offs that are regularly used to determine tumor MGMT status in the clinic, an international survey was provided to those working in the field. We also inquired about opinions regarding an international consensus on how MGMT should be tested. The 152 respondents reported several methodologies and different cut-off levels also for the same method. A majority of respondents warrant international guidelines.In conclusion, the results of the 2 randomized trials contribute to individualized treatment recommendations for patients affected by GBM or astrocytoma grade III. The results of the survey regarding analyses of MGMT clarify the current problematic situation. The request of the respondents regarding international guidelines might contribute to their future development, so that personalized treatment recommendations can be improved.
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| 2. |
- Olsson Lindvall, Martina, et al.
(författare)
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Comparison of DNA Methylation Profiles of Hemostatic Genes between Liver Tissue and Peripheral Blood within Individuals
- 2021
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Ingår i: Thrombosis and Haemostasis. - : Georg Thieme Verlag KG. - 0340-6245 .- 2567-689X. ; 121:5, s. 573-583
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Tidskriftsartikel (refereegranskat)abstract
- DNA methylation has become increasingly recognized in the etiology of complex diseases, including thrombotic disorders. Blood is often collected in epidemiological studies for genotyping and has recently also been used to examine DNA methylation in epigenome-wide association studies. DNA methylation patterns are often tissue-specific, thus, peripheral blood may not accurately reflect the methylation pattern in the tissue of relevance. Here, we collected paired liver and blood samples concurrently from 27 individuals undergoing liver surgery. We performed targeted bisulfite sequencing for a set of 35 hemostatic genes primarily expressed in liver to analyze DNA methylation levels of >10,000 cytosine-phosphate-guanine (CpG) dinucleotides. We evaluated whether DNA methylation in blood could serve as a proxy for DNA methylation in liver at individual CpGs. Approximately 30% of CpGs were nonvariable and were predominantly hypo- (<25%) or hypermethylated (>70%) in both tissues. While blood can serve as a proxy for liver at these CpGs, the low variability renders these unlikely to explain phenotypic differences. We therefore focused on CpG sites with variable methylation levels in liver. The level of blood-liver tissue correlation varied widely across these variable CpGs; moderate correlations (0.5 <= r <0.75) were detected for 6% and strong correlations ( r 0.75) for a further 4%. Our findings indicate that it is essential to study the concordance of DNA methylation between blood and liver at individual CpGs. This paired blood-liver dataset is intended as a resource to aid interpretation of blood-based DNA methylation results.
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| 3. |
- Karlsson, Iulia
(författare)
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Cytokines as diagnostic biomarkers in canine pyometra and sepsis
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Sepsis is a syndrome with high morbidity, mortality and astronomical health care costs and it is challenging to diagnose both in humans and animals due to the lack of suitable diagnostic biomarkers. Although several types of proteins have been suggested as diagnostic biomarkers of sepsis, none of them were shown to be reliable for routine use in the clinical practice. Dogs with uterine bacterial infection called pyometra often develop sepsis and have been suggested as a natural model of sepsis. To investigate whether there is a pattern of biomarkers that can be useful to diagnose bacterial sepsis on early stages in addition to existing clinical criteria, we measured both local gene expression and serum levels of cytokines in dogs with pyometra and compared these levels with known inflammatory markers and blood clotting parameters. Serum concentrations of keratinocyte-derived chemokine (KC)-like protein and the global clot strength were significantly increased both in dogs with pyometra compared to healthy dogs and in dogs with sepsis compared to dogs without sepsis in pyometra. Moreover, the expression levels of the chemokines interleukin (IL)-8 and C-X-C motif ligand 5 (CXCL5) mRNA were significantly higher in uteri from dogs with pyometra compared to healthy dogs and in cultured stromal endometrial cells derived from uteri of healthy dogs and cocultured with LPS or pathogenic Escherichia coli compared to unstimulated cells. Although serum concentrations of IL-8, high-mobility group box 1 (HMGB1), prostaglandin F2α, IL-2, IL-15, IL-18, interferon (IFN)-γ and monocyte-macrophage colony stimulating factor (MG-CSF) were not different between dogs with or without sepsis in the presence of pyometra, some of these cytokines correlated significantly with clinical parameters such as total white blood cell count (correlated with HMGB1) and KC-like (correlated with IL-8). Measurements of serum IL-10, CXCL10, tumor necrosis factor (TNF)-α, IL-6 and IL-4 will require a more sensitive method in dogs with pyometra. Our findings suggest that KC-like, CXCL5 and IL-8 may be useful as early diagnostic biomarkers of sepsis in dogs with pyometra. Further investigation of these chemokines in sepsis may help to improve routines in sepsis diagnosis in dogs and possibly also humans.
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| 4. |
- Bergman, Daniel, et al.
(författare)
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Investigation of interference from canine anti-mouse antibodies in hormone immunoassays
- 2019
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Ingår i: Veterinary clinical pathology. - : Wiley. - 0275-6382 .- 1939-165X. ; 48:S1, s. 59-69
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Canine anti-mouse antibodies are a potential source of immunoassay interference, but erroneous immunoassay results are not always easily identifiable. Anti-Müllerian hormone (AMH) is a marker for the presence of gonads in dogs, but elevated AMH concentrations in neutered dogs could also be caused by antibody interference. For other assays, a discrepant result obtained after antibody precipitation might indicate antibody interference.OBJECTIVES: We aimed to evaluate if canine anti-mouse antibodies are a source of erroneous results in the AMH assay and if antibody precipitation with polyethylene glycol (PEG) is a useful tool for detecting antibody interference in a variety of immunoassays used in the veterinary clinical laboratory.METHODS: Twenty-nine positive and 25 negative samples for anti-mouse antibodies were analyzed for AMH, canine total thyroxine (TT4), canine thyroid-stimulating hormone (TSH) and progesterone before and after treatment with PEG. Results that differed by more than four SDs from the intra-assay coefficients of variation were considered discrepant. Elevated AMH concentrations in neutered dogs with anti-mouse antibodies and no visible gonads present were considered evidence of interference.RESULTS: Evidence of antibody interference was found in two samples analyzed for AMH. The presence of anti-mouse antibodies did not lead to a higher proportion of discrepant results after PEG treatment for any of the immunoassays. The overall incidence of discrepant results for healthy controls was very high (73%).CONCLUSIONS: Canine anti-mouse antibodies are a source of erroneous AMH results. Antibody precipitation with PEG is not a useful tool for detecting interference caused by such antibodies.
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| 5. |
- Malmström, Annika, 1957-, et al.
(författare)
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Do we really know who has an MGMT methylated glioma? : results of an international survey regarding use of MGMT analyses for glioma
- 2020
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Ingår i: Neuro-Oncology Practice. - Oxford : Oxford University Press. - 2054-2577 .- 2054-2585. ; 7:1, s. 68-76
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Tidskriftsartikel (refereegranskat)abstract
- Background: Glioma O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status informs clinical decision making. Worldwide different methods and cutoff levels are used, which can lead to discordant methylation results.Methods: We conducted an international survey to clarify which methods are regularly used and why. We also explored opinions regarding international consensus on methods and cutoff.Results: The survey had 152 respondents from 25 countries. MGMT methylation status is determined for all glioblastomas in 37% of laboratories. The most common methods are methylation-specific polymerase chain reaction (msPCR) (37%) and pyrosequencing (34%). A method is selected for simplicity (56%), cost-effectiveness (50%), and reproducibility of results (52%). For sequencing, the number of CpG sites analyzed varies from 1–3 up to more than 16. For 50% of laboratories, the company producing the kit determines which CpG sites are examined, whereas 33% select the sites themselves. Selection of cutoff is equally distributed among a cutoff defined in the literature, by the local laboratory, or by the outside laboratory performing the analysis. This cutoff varies, reported from 1% to 30%, and in 1 laboratory tumor is determined as methylated in case of 1 methylated CpG site of 17 analyzed. Some report tumors as unmethylated or weakly vs highly methylated. An international consensus on MGMT methylation method and cutoff is warranted by 66% and 76% of respondents, respectively. The method preferred would be msPCR (45%) or pyrosequencing (42%), whereas 18% suggest next-generation sequencing.Conclusion: Although analysis of MGMT methylation status is routine, there is controversy regarding laboratory methods and cutoff level. Most respondents favor development of international consensus guidelines.
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| 6. |
- Nordin, Elise, 1985, et al.
(författare)
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An inverse association between plasma benzoxazinoid metabolites and PSA after rye intake in men with prostate cancer revealed with a new method
- 2022
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Prostate cancer (PC) is a common cancer among men, and preventive strategies are warranted. Benzoxazinoids (BXs) in rye have shown potential against PC in vitro but human studies are lacking. The aim was to establish a quantitative method for analysis of BXs and investigate their plasma levels after a whole grain/bran rye vs refined wheat intervention, as well as exploring their association with PSA, in men with PC. A quantitative method for analysis of 22 BXs, including novel metabolites identified by mass spectrometry and NMR, was established, and applied to plasma samples from a randomized crossover study where patients with indolent PC (n = 17) consumed 485 g whole grain rye/rye bran or fiber supplemented refined wheat daily for 6 wk. Most BXs were significantly higher in plasma after rye (0.3–19.4 nmol/L in plasma) vs. refined wheat (0.05–2.9 nmol/L) intake. HBOA-glc, 2-HHPAA, HBOA-glcA, 2-HPAA-glcA were inversely correlated to PSA in plasma (p < 0.04). To conclude, BXs in plasma, including metabolites not previously analyzed, were quantified. BX metabolites were significantly higher after rye vs refined wheat consumption. Four BX-related metabolites were inversely associated with PSA, which merits further investigation.
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| 7. |
- Tvedten, Harold, et al.
(författare)
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Validation of Advia plateletcrit for assessing platelet mass in dogs, including Cavalier King Charles Spaniels
- 2012
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Ingår i: Veterinary Clinical Pathology. - 0275-6382 .- 1939-165X. ; 41, s. 336-343
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Tidskriftsartikel (refereegranskat)abstract
- Background Determination of the plateletcrit (PCT) is the most effective way to evaluate platelet mass in dogs, such as Cavalier King Charles spaniel (CKCS) dogs, with macrothrombocytopenia. The IDEXX VetAutoread hematology analyzer, which performs quantitative buffy coat (QBC) analysis, has been validated to determine platelet mass in CKCS dogs. The Advia 2120 reports a PCT, but the validity of this value has not been evaluated for dogs with macrothrombocytopenia. Objectives The goal of this study was to validate MPV and PCT determined by the Advia 2120 in dogs, including CKCS dogs, comparing values with those obtained from QBC analysis. Methods Advia PCT was compared with QBC results from 43 CKCS dogs and 15 dogs of other breeds in one study. Advia PCT, platelet count, and MPV were evaluated to identify biologic patterns in 31 clinically healthy CKCS dogs and 66 dogs of 3 other breeds and to generate values used for comparisons. Results Advia PCT agreed well with QBC results in general, but had a negative bias and appeared to underestimate PCT in CKCS dogs with the lowest PCTs. Advia PCT and MPV results followed expected biologic patterns in CKCS dogs and dogs of other breeds with MPVs being highest in dogs with the lowest platelet counts. Conclusions Advia 2120 PCT and MPV satisfactorily identified changes in platelet mass and size in CKCS dogs, but PCTs were lower than expected, especially in CKCS dogs with the lowest PCTs, when compared with QBC results.
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| 8. |
- Bergman, Daniel
(författare)
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Canine heterophilic antibodies
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Anamnesis, physical examination and laboratory testing are the pillars of the clinical diagnostic procedure. Alas, laboratory tests are not perfect and analytical errors happen, which can lead to misdiagnosis and detrimental consequences for patient care. Immunoassays are commonly used to measure various hormones and disease markers in patient samples. Despite decades of methodological development and technological advances, immunoassays used for clinical diagnosis are still associated with limitations and even some flaws.This thesis focuses on a long-lived immunoassay flaw that has been poorly researched in veterinary medicine. Humans and animals both carry heterophilic antibodies, also called anti-animal antibodies, in their circulation. These antibodies can interfere with immunoassays and cause erroneous results. The mechanism of action is the same for animals as it is for humans; the heterophilic antibodies bind to animal antibodies employed by the immunoassay, usually leading to a falsely increased measurement. Due to the extensive use of mouse IgG for analyte detection in immunoassays, anti-mouse antibodies are of particular concern.Herein, the prevalence of heterophilic antibodies against mouse IgG in a cohort of dog patients is estimated. It is demonstrated that the antibodies can have tangible consequences for patient care as they can interfere with commercial immunoassays used in veterinary laboratories. Falsely increased anti-Müllerian hormone (AMH) measurements were found, which could lead to needless surgery in dog patients. The molecular characteristics of canine heterophilic antibodies were shown to be heterogeneous. They may react with the Fc region or the Fab region of the murine IgG molecule. There is cross-reactivity with IgG from several species, and heterophilic antibodies in dogs are made up of the IgA, IgG and IgM isotypes. The prevalence of the antibodies varies between dog breeds, and the Bernese mountain dog is tentatively predisposed to heterophilic antibodies. The origin of these antibodies remains mostly unclear, but there is occasional cross-reactivity between antibodies to mouse IgG and canine autoantibodies to IgG. Canine heterophilic antibodies can persist for at least two years in serum and represent a risk factor for repeated analytical errors and misdiagnosis in patients with these antibodies.
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| 9. |
- Bergman, Daniel, et al.
(författare)
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Pre-existing canine anti-IgG antibodies: implications for immunotherapy, immunogenicity testing and immunoassay analysis
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- One of the most enigmatic features of humoral immunity is the prevalent presence of circulating autoantibodies against IgG. These autoantibodies consist of several subsets, including rheumatoid factors, anti-Fab/anti-F(ab′)2-autoantibodies, and anti-idiotypic antibodies. Anti-IgG autoantibodies can impair the safety and efficacy of therapeutic antibodies and interfere with immunogenicity tests in clinical trials. They can also cross-react with allospecific IgG, presenting as heterophilic antibodies that interfere with diagnostic immunoassays. Owing to these factors, recent years have seen a resurgent interest in anti-IgG autoantibodies, but their underlying clinical significance, as well as biological roles and origins, remain opaque. Increased knowledge about canine anti-IgG autoantibodies could facilitate the development of canine immunotherapies and help in understanding and counteracting immunoassay interference. This study investigated the clinical significance and interconnection of heterophilic antibodies, anti-Fab, and anti-F(ab′)2-autoantibodies in dogs. We performed a 2-year prospective follow-up of dogs with heterophilic antibodies and analyzed serum for anti-Fab and anti-F(ab′)2-autoantibodies. Canine heterophilic antibodies can persist for at least 2 years in serum. A widespread occurrence of anti-Fab and anti-F(ab′)2-autoantibodies was found, with reactivity to cryptic epitopes in the IgG hinge region and sporadic cross-reactivity with mouse IgG. Canine anti-Fab and anti-F(ab′)2-autoantibodies are thus potential sources of clinical immunogenicity and immunoassay interference.
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| 10. |
- Hillström, Anna
(författare)
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Canine C-reactive protein : validation of two automated canine-specific C-reactive protein assays and studies on clinical and research applications
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- C-reactive protein (CRP) is a sensitive and specific marker of systemic inflammation in dogs, valuable for diagnosing and monitoring inflammatory diseases. The use of CRP in canine medicine has however been hampered by the lack of automated assays optimized for measuring CRP in this species. The need for improved CRP assays was the reason for initiating the current project, with the goal to generate automated, canine-specific CRP tests that could reliably measure serum CRP over the whole concentration range expected to occur in dogs. Two different assays were developed for this purpose. One was designed for routine diagnostic testing, and one was a high-sensitivity CRP test intended for research. Method validation studies were performed, demonstrating that both tests met the predefined quality criteria. Using the two novel CRP tests, it was possible to reliably measure serum CRP concentrations in the range of 0.5-1200 mg/l. After successful termination of the validation studies, the CRP assays were used in clinical research studies. C-reactive protein concentrations were measured in dogs with pyometra undergoing ovariohysterectomy, to evaluate how surgical treatment affected degree of systemic inflammation in these patients. Two other studies were performed to evaluate the usefulness of CRP as a diagnostic test. C-reactive protein concentration was found to discriminate well between dogs with suppurative arthritis and dogs with osteoarthritis, whereas measurement of CRP was not efficient for diagnosing late post-operative bacterial infections after orthopaedic surgery because these infections often did not elicit a systemic inflammatory response. In conclusion, two novel automated canine-specific CRP assays were developed and validated with satisfactory results. The tests showed high practicability for measuring CRP in samples from clinical research studies. Availability of these assays will facilitate the use of CRP as a routine diagnostic test in veterinary medicine, and can improve quality in research on canine inflammatory diseases.
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