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Hypogammaglobulinaemia during rituximab treatment in multiple sclerosis: A Swedish cohort study.
No full textBACKGROUND AND PURPOSE: Mechanisms behind hypogammaglobulinaemia during rituximab treatment are poorly understood. METHODS: In this register-based multi-centre retrospective cohort study of multiple sclerosis (MS) patients in Sweden, 2745 patients from six participating Swedish MS centres were identified via the Swedish MS registry and included between 14 March 2008 and 25 January 2021. The exposure was treatment with at least one dose of rituximab for MS or clinically isolated syndrome, including data on treatment duration and doses. The degree of yearly decrease in immunoglobulin G (IgG) and immunoglobulin M (IgM) levels was evaluated. RESULTS: The mean decrease in IgG was 0.27 (95% confidence interval 0.17-0.36) g/L per year on rituximab treatment, slightly less in older patients, and without significant difference between sexes. IgG or IgM below the lower limit of normal (</p
Prioritization of mechanisms and potential biomarkers in inflammatory and malignant diseases
No full textDespite significant advancements in biomedical research, diseases such as inflammatory and malignant conditions remain challenging to diagnose and treat effectively. These diseases exhibit shared underlying complexities, including the involvement of thousands of genes across various organs and cell types. This complexity hinders the identification and prioritization of reliable biomarkers and drug targets. Single-cell RNA sequencing (scRNA-seq) and multi-omics analyses have emerged as powerful tools to address these challenges. Specifically, analyzing cell-cell communication (CCC) using scRNA-seq data enables prioritization of upstream regulators (URs), which represent higher-order molecular mechanisms and may serve as key drivers in various diseases. However, systematic approaches are needed to identify and validate these URs as clinically relevant biomarkers.The overarching aim of this PhD thesis was to systematically prioritize regulatory mechanisms in inflammatory and malignant diseases. By analyzing multi-omics data, including scRNA-seq, spatial transcriptomics, proteomics, and clinical datasets, we aimed to: 1) characterize complex gene expression changes using transcriptional programs and URs in inflammatory diseases; 2) extend this approach to prioritize biomarkers in pancreatic cancer; 3) identify URs shared across multiple cancer types; and 4) evaluate the clinical utility of protein biomarkers in tissue and blood for cancer diagnosis.In this PhD thesis, we demonstrated that higher-order structures (transcriptional programs and URs) could be identified in immune-mediated inflammatory diseases (IMIDs), revealing a graded on/off system in immune regulation across multiple organs (Study I). These higher-order structures could be extended to pancreatic cancer, uncovering URs associated with two transcriptional programs linked to cancer risk and severity (Study II). Further analysis in Study III identified shared URs (shared-URs) across various cancer types that were associated with survival outcomes. This significant association of the prioritized URs with clinical traits including risk of disease and survival in large independent cohorts supported the general relevance of this approach. Moreover, Study IV revealed high diagnostic accuracy for tissue-derived proteins but limited predictive value for blood-based biomarkers using a machine learning approach.In conclusion, this thesis offers insights into disease mechanisms and potential biomarkers for diagnosis and prognosis, utilizing both systematic computational analyses based on biological knowledge and machine-learning (ML) approaches. These approaches may contribute to developing a scalable framework to identify biomarkers and drug targets in inflammatory and malignant diseases.List of scientific papersI. Sandra Lilja*, Xinxiu Li*, Martin Smelik*, Eun Jung Lee, Joseph Loscalzo, Pratheek Bellur Marthanda, Lang Hu, Mattias Magnusson, Oleg Sysoev, Huan Zhang, Yelin Zhao, Christopher Sjöwall, Danuta Gawel, Hui Wang, Mikael Benson. Multi-organ single-cell analysis reveals an on/off switch system with potential for personalized treatment of immunological diseases. Cell Rep Med. 21;4(3):100956 (2023). https://doi.org/10.1016/j.xcrm.2023.100956II. Yelin Zhao, Martin Smelik, Xiaojing An, Daniel Diaz-Roncero Gonzalez, Xinxiu Li, AKM Firoj Mahmud, Oleg Sysoev, Dina Mansour Aly, Mikael Benson. Single-cell and Spatial Transcriptomics Reveal Fibroblast Related Program Linked to Pancreatic Cancer Risk in the UK Biobank Cohort. [Manuscript]III. Yelin Zhao, Xinxiu Li, Joseph Loscalzo, Martin Smelik, Oleg Sysoev, Yunzhang Wang, A. K. M. Firoj Mahmud, Dina Mansour Aly, Mikael Benson. Transcript and protein signatures derived from shared molecular interactions across cancers are associated with mortality. J Transl Med 22, 444 (2024). https://doi.org/10.1186/s12967-024-05268-7IV. Martin Smelik, Yelin Zhao, Dina Mansour Aly, AKM Firoj Mahmud, Oleg Sysoev, Xinxiu Li, Mikael Benson. Multiomics biomarkers were not superior to clinical variables for pan-cancer screening. Communications Medicine. [Accepted]*Shared first-author</p
Targeting ischemia-reperfusion injury in kidney transplantation
No full textFor patients with end-stage renal disease, kidney transplantation represents an effective treatment option, providing substantial improvements in quality of life and cost-effectiveness. Although transplantation outcomes are generally favorable, there is potential for further improvement, particularly by reducing early damage to the transplanted kidney, which significantly impacts long-term graft survival. Considering ongoing organ shortages, and extended criteria for deceased donors, a better understanding of early post-transplant events, particularly ischemia-reperfusion injury (IRI), is important for optimizing patient outcomes.This thesis explores different aspects of IRI, an unavoidable consequence of organ donation and transplantation, through several experimental and clinical approaches. The aim has been to increase our understanding of the mechanisms behind IRI in kidney transplantation and to evaluate different strategies to mitigate this condition.Study I investigated the effects of a novel oxygenated organ preservation solution in a porcine model of kidney transplantation from brain-dead donors. The findings indicate that this oxygenated preservation solution may offer benefits over a standard, non-oxygenated preservation solution in reducing IRI. Given that IRI is a critical factor influencing graft function and survival, these results suggest that advancements in organ preservation techniques, such as oxygenation during cold storage, could improve outcomes in kidney transplantation.Study II examined the early molecular response to IRI during living donor kidney transplantation, and whether remote ischemic preconditioning (RIPC) of the donors could modulate this response. The study was divided in three parts: First an analysis of early gene expression associated with IRI in living donor kidney transplantation, which revealed pronounced and significant alterations. Secondly, the study explored the effect of RIPC, to assess whether this intervention could modulate early gene expression associated with IRI, where no significant differences in gene expression were observed between treated and untreated groups. Finally, a comparative pathway analysis, including the kynurenine pathway (KYNA-P), was conducted to compare findings from previously published studies in animal models with the human data obtained in this study. This analysis indicated that some pathways activated in animals were not activated in human donors or healthy volunteers treated with RIPC. Although the RIPC protocol used in this study is commonly applied and has demonstrated effects in other settings, it did not yield detectable effects in our specific context, which focused on investigating early gene expression and measuring metabolites of the KYNA-P.Study III, focused on comparing two experimental models of IRI, one conducted in vivo, and the other ex vivo. In a porcine model of donation after circulatory death followed by kidney transplantation, the effects of IRI on renal allograft gene expression and histopathology were analyzed and compared to those in an ex vivo precision-cut kidney slices (PCKS) cell culture model. No difference in gene expression was observed between models proving that our ex vivo model can be used to a certain extent instead of the kidney transplant model in vivo. The absence of circulating blood and immune cells in the ex vivo model limited its ability to fully replicate the in vivo response to IRI.Study IV was a clinical phase 1b pharmaceutical trial involving eight recipients of kidneys from living donors, evaluating the pharmacokinetics and safety profile of the study drug, RMC-035, a modified form of the endogenous protein alpha-1- microglobulin, with potential protective effects against IRI. The results demonstrated dose-linearity, with the drug well tolerated by the participants and no significant differences from the typically favorable outcomes observed in standard living donor kidney transplantation. Deeper investigation is necessary to investigate RMC 035's potential protective effects on IRI.In summary, this thesis explores several aspects of IRI in kidney transplantation, identifying the potential benefits of an oxygenated preservation solution, and illustrates the need to carefully evaluate new potentially protective interventions and pharmacological drugs. As also shown in this thesis, IRI experiments in ex vivo models can be informative and useful. Possibly, the thesis has also contributed to an increased understanding of IRI. However, continued studies on the mechanisms behind IRI and potential modalities to mitigate IRI, are needed.List of scientific papersI. Asif S, Sedigh A Nordstrom J*, Brandhorst H, Jorns C, Lorant T, Larsson E, Magnusson PU, Nowak G, Theisinger S, Hoeger S, Wennberg L, Korsgren O, Brandhorst D. Oxygen-charged HTK-F6H8 emulsion reduces ischemia-reperfusion injury in kidney from brain- dead pigs. Journal of Surgical Research. 2012 December;178(2):959-967. https://doi.org/10.1016/j.jss.2012.06.031 *2nd authorII. Nordström J*, Badia-I-Mompel P*, Witasp A, Schwarz A, Evenepoel P, Moor MB, Wennberg L, Saez-Rodriguez J, Wernerson A, Olauson H. Defining the molecular response to ischemia-reperfusion injury and remote ischemic preconditioning in human kidney transplantation. PLoS ONE 2024 October;19(10):e0311613. https://doi.org/10.1371/journal.pone.0311613 *Shared 1st author III. Moor M*, Nordström J*, Burmakin M, Raki M, Al-Saad S, Nowak G, Wennberg L, Patrakka J, Olauson H. Comparative analysis of kidney transplantation modeled using precision-cut kidney slices and kidney transplantation in pigs. [Manuscript] *Shared 1st authorIV. Nordström J*, Wennberg L, Nowak G, Larsson T, Thuresson S, Reusch M. Assessing Pharmacokinetics and Safety of Therapeutic Alpha-1-Microglobulin in First-in-Human Kidney Transplantation: A Non-Comparative Open Label Multiple Dose Phase 1b Study. Transplantation Direct 2024 December;10(12):p e1727. https://doi.org/10.1097/TXD.0000000000001727 *1st author</p
MAIT cell heterogeneity across paired human tissues reveals specialization of distinct regulatory and enhanced effector profiles.
No full textMucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize microbial riboflavin pathway metabolites presented by evolutionarily conserved MR1 molecules. We explored the human MAIT cell compartment across organ donor-matched blood, barrier, and lymphoid tissues. MAIT cell population size was donor dependent with distinct tissue compartmentalization patterns and adaptations: Intestinal CD103+ resident MAIT cells presented an immunoregulatory CD39highCD27low profile, whereas MAIT cells expressing NCAM1/CD56 dominated in the liver and exhibited enhanced effector capacity with elevated response magnitude and polyfunctionality. Both intestinal CD39high and hepatic CD56+ adaptations accumulated with donor age. CD56+ MAIT cells displayed limited T cell receptor-repertoire breadth, elevated MR1 binding, and a transcriptional profile skewed toward innate activation pathways. Furthermore, CD56 was dynamically up-regulated to a persistent steady-state equilibrium after exposure to antigen or IL-7. In summary, we demonstrate functional heterogeneity and tissue site adaptation in resident MAIT cells across human barrier tissues with distinct regulatory and effector signatures
ECAS correlation with metabolic alterations on FDG-PET imaging in ALS.
No full textBackground: Cognitive impairment is observed in up to 50% of patients with amyotrophic lateral sclerosis (ALS). The Edinburgh Cognitive and Behavioral ALS Screen (ECAS) is an ALS-specific multi-domain screening tool. Few studies have examined the relationship between ECAS scores and [18F]fluorodeoxyglucose positron emission tomography ([18F]FDG-PET) findings. Objective: To assess the relationship between ECAS scores and glucose metabolism patterns on [18F]FDG -PET images in ALS. Methods: We collected [18F]FDG-PET images from 65 patients with ALS and 39 healthy controls. ECAS scores were collected on all patients and we calculated the correlation to [18F]FDG-PET in order to investigate the potential links between cognition and glucose metabolism. Results: We observed hypometabolism in the frontal cortex, insula, and limbic system, together with hypermetabolism in the cerebellum in patients with ALS compared to controls. A lower ECAS total score was associated with lower glucose metabolism in the right orbitofrontal gyrus and higher glucose metabolism in lateral occipital, medial occipital, and cerebellar regions, among patients with ALS. Similar results, although less widespread, were observed in the analyses of ECAS ALS-specific scores. Conclusions: The metabolic patterns in [18F]FDG -PET show that changes in the glucose metabolism of corresponding areas are related to cognitive dysfunction in ALS, and can be detected using the ECAS
Molecular prognostication of thyroid tumors : with special focus on TERT
No full textThyroid carcinoma is the most common endocrine malignancy, with its incidence steadily increasing worldwide. These tumors can be challenging to diagnose, and treatment decisions are typically based on tumor size and a limited number of histomorphological features. To improve diagnosis, risk assessment, and treatment outcomes, there has been a growing focus on identifying new markers. In other types of tumors, molecular and immunohistochemical markers have significantly enhanced the diagnostic accuracy and prognostic evaluation. However, research on thyroid tumors has not yet resulted in markers that affect the management guidelines for thyroid tumors. Several potentially important markers are yet to be examined in terms of their effect on patient outcomes. My thesis aimed to study the clinical implication of molecular and immunohistochemical markers in thyroid tumors, with a special focus on telomerase reverse transcriptase (TERT) promoter mutations.In Paper I, we examined the Ki-67 labeling index and its diagnostic and prognostic value in follicular thyroid tumors. Ki-67 is a nuclear protein expressed during all phases of the cell cycle except the resting state. Immunohistochemical analysis of Ki-67 is a widely used tool for assessing cell proliferation in various tumor types. Although it is used in the assessment of thyroid tumors, it is not determined exactly when or how to use it. In our study, we found that a Ki-67 index >4% was the most optimal threshold for indicating malignancy. However, there was considerable overlap between benign and malignant tumors using this cut-off, limiting its utility as a purely diagnostic marker. On the other hand, a Ki- 67 labeling index >4% effectively identified patients with follicular thyroid carcinoma (FTC) at risk of recurrence with metastatic disease. This cut-off allowed us to predict recurrence in FTCs more accurately, both across the entire group and within individual tumor stage subgroups. Moreover, the Ki-67 labeling index was an independent predictor of both metastatic disease and disease- specific mortality, suggesting its potential as an important prognostic tool in thyroid tumors. Although these results need to be reproduced in additional studies, our research demonstrates the potential of Ki-67 as a valuable marker in the evaluation of follicular thyroid tumors. To date, this is the largest study examining Ki-67 data in follicular thyroid tumors.In Paper II, we examined if TERT promoter mutational analysis, using digital droplet polymerase chain reaction (ddPCR), could be performed on preoperative fine-needle aspiration cytology (FNAC) samples. We compared the preoperative mutational status of tumors with their postoperative status from the same tumors. Our results demonstrated a perfect correlation between the preoperative FNAC and postoperative formalin-fixed paraffin-embedded (FFPE) samples, with both mutated and wild-type tumors retaining their status in both contexts. Notably, all tumors with TERT promoter mutations were malignant, highlighting the potential of preoperative mutational analysis as a diagnostic tool for malignancy. Additionally, these mutations were associated with more aggressive histological subtypes, further underscoring their prognostic significance in the preoperative setting. In conclusion, ddPCR proved highly effective in detecting TERT promoter mutations in FNAC samples, showing high specificity. If validated in larger studies, this approach could influence surgical decisions for a subset of indeterminate thyroid lesions.In Paper III, we introduced a novel in situ hybridization (ISH) technique to analyze TERT mRNA expression. TERT expression is associated with poor prognosis in FTCs, often due to TERT promoter mutations, but can also result from gene amplification or promoter hypermethylation. This novel technique allows for the visualization of TERT mRNA expression directly within the tissue, regardless of the underlying cause. We tested this method on FTCs with known TERT mRNA expression and TERT promoter mutation status, using two separate probes targeting different regions of the mRNA sequence. Our findings revealed that eight out of ten cases with known expression were detected by at least one of the probes, with no signals observed in TERT mRNA-negative samples. Interestingly, the positive signals were confined to the nuclear compartment of tumor cells and appeared in only a small fraction of the cells. This nuclear localization suggests that TERT mRNA may have an unconventional role in thyroid cancer development, warranting further investigation. Given the accuracy of ISH in identifying TERT promoter-mutated cases with confirmed mRNA expression, it holds significant potential for clinical screening of TERT abnormalities.In Paper IV, we evaluated a clinical screening program designed to detect TERT promoter mutations in minimally invasive follicular thyroid carcinoma (miFTC) and minimally invasive oncocytic thyroid carcinoma (miOTC) using ddPCR. We analyzed a cohort of patients diagnosed with miFTC or miOTC. TERT promoter mutations were detected in 11.9% of the cases, including a small pT1 tumor measuring less than 20 mm. The mutations were frequently subclonal, and patients with mutated tumors were generally older compared to those with wild-type tumors. However, other clinical variables, such as tumor size and metastatic rate, did not differ significantly between the mutated and wild-type groups. These findings suggest that TERT promoter mutations can occur in miFTCs and miOTCs regardless of tumor size, supporting the rationale for testing all such tumors, regardless of tumor stage. The short follow-up time, along with the fact that the mutations were often subclonal, additional studies with longer follow-up periods are needed to fully determine their clinical relevance.List of scientific papersI. Hellgren LS, Stenman A, Paulsson JO, Höög A, Larsson C, Zedenius J, Juhlin CC. Prognostic utility of the Ki-67 labeling index in follicular thyroid tumors: a 20-year experience from a tertiary thyroid center. Endocr Pathol. 2022 Jun;33(2):231-42. https://doi.org/10.1007/s12022-022-09714-4II. Hysek M, Hellgren LS, Stenman A, Darai-Ramqvist E, Ljung E, Schliemann I, Condello V, Larsson C, Zedenius J, Juhlin CC. Digital droplet PCR TERT promoter mutational screening in fine needle aspiration cytology of thyroid lesions: A highly specific technique for pre-operative identification of high-risk cases. Diagn Cytopathol. 2023 Jun;51(6):331-40. https://doi.org/10.1002/dc.25120III. Hellgren LS, Olsson A, Kaufeldt A, Paulsson JO, Hysek M, Stenman A, Zedenius J, Larsson C, Juhlin CC. Nuclear-specific accumulation of telomerase reverse transcriptase (TERT) mRNA in TERT promoter mutated follicular thyroid tumours visualised by in situ hybridisation: a possible clinical screening tool? J Clin Pathol. 2021 May 19;75(10):658-62. https://doi.org/10.1136/jclinpath-2021-207631IV. Hellgren LS, Stenman A, Jatta K, Condello V, Larsson C, Zedenius J, Juhlin CC. Catching the silent culprits: TERT promoter mutation screening of minimally invasive follicular and oncocytic thyroid carcinoma in clinical practice. Endocr Pathol. 2024 Oct 4. Online ahead of print. https://doi.org/10.1007/s12022-024-09828-x</p
Hip surgery to prevent hip dislocation in children with cerebral palsy
No full textBackground: Cerebral palsy (CP) refers to a group of disorders that primarily affect movement, muscle tone, and posture. It is caused by abnormal brain development or damage to the developing brain with an incidence of 2 to 3 per 1,000 births. The most affected children with CP (non-ambulatory) have a high risk (up to 90%) of hip dislocation. In Sweden, the Cerebral Pares UppföljningsProgram (CPUP) is a National Quality Register that includes all children diagnosed with CP born from 2003 onwards, involving around 4,000 children. The great advantage of this type of register is its extent, allowing the study of the entire population of children with CP. The primary purpose of this thesis was to study the outcome after hip surgery and a summary of different orthopedical surgical procedures.Methods: All studies of this thesis are based on registry data from CPUP. In Study II, data from the National Patient Registry (NPR) were also used, and in Study IV, CPUP-data from only two regions were included. The data extracted from the CPUP register included age at operation and reoperation, gender, Gross Motor Function Classification System (GMFCS) level, CP subtype, and the radiographic variables Migration Percentage (MP), Acetabular Index (AI), and Head-Shaft Angle (HSA).In study I we identified 186 children with CP who underwent either adductor/iliopsoas tenotomy (APT) or femoral osteotomy (FO) as the primary preventive surgery due to hip displacement. The cohort was followed for five years, regarding the hip migration (MP) and the need for revision surgery. Outcome failure was defined as either repeat surgery or MP>50%.In the second study, using data from the CPUP and NPR registers, we followed 3,305 children and young adults to describe the risk of undergoing primary orthopedic surgical treatment in relation to age.In study III we identified and followed for five years all 163 children who had been treated with FO or with combined femur and pelvic osteotomy (FPO) in the CPUP register, regarding reoperation rates or MP >50%, using the same definition of outcome failure as in study I In the fourth study, we described and analyzed the 5-year radiological follow-up regarding MP, AI, and HSA in 72 children who underwent reconstructive hip surgery with FO and/or FPO in two regions in Sweden.Results: Out of the 129 children who had had adductor-iliopsoas tenotomy (APT), 56 (43%) had repeated surgery, and 2 children (2%) with MP>50% at the 5-year follow-up. Out of the 57 children who had had at least one proximal femur osteotomy with or without concomitant pelvic osteotomy (FO) as primary surgery, 22 children (39%) were reoperated within 5 years (24 hips, 30% of all hips) (Study I).In study II, data for 3,311 orthopedic surgeries performed during 1,717 surgery sessions were collected. The percentage of children undergoing surgery before age 15 increased from 20% in GMFCS level I to 64% in level. Surgeries at the foot and lower leg level, dominated in GMFCS I and II. Hip and femur surgery was most common in GMFCS level IV or V, as was spine surgery. Repeated surgeries were more common for higher GMFCS levels.In study III, two types of skeletal corrections were compared. Out of 63 combined proximal femur and pelvic osteotomies (FPO), only 2 hips were re- operated, and four hips had an MP>50% at the last follow-up. Out of 124 isolated proximal femur osteotomies FO, 25 hips had repeated orthopedic surgery, and 16 hips had an MP>50% at the last follow-up. Multivariate analysis showed that FPO reduced the risk of treatment failure by more than 60%.In 72 children with radiological 5-year follow-up who underwent 103 reconstructive surgeries (FO or FPO), there was a statistically significant difference regarding only the AI parameter at the end of the follow-up (Study IV).Conclusions: The high reoperation rates within 5 years after APT and FO as primary surgical treatment for hip displacement emphasizes the importance of continuous postoperative follow-up. The percentage of young individuals undergoing orthopedic surgery increased as GMFCS levels rose. Among ambulatory children and adolescents (GMFCS I), around 20% had undergone a surgical intervention before age 15 years, commonly involving the lower leg or foot, such as Achilles tendon lengthening. Among children needing support to ambulate (GMFCS level II or III), up to 30-35% had undergone similar surgery. For children reliant on a wheelchair for transport (GMFCS level IV or V), surgery at the hip and spine was more common. Approximately one-fourth of the children at GMFCS level V had undergone hip surgery before the age of 5, and about 50% of the children by the age of 12.In the comparison between treatment with FO or FPO, the proportion of re- operated hips within five years was considerably lower for FPO than for FO. FPO lowered the risk of failure by more than 60% compared with FO. Despite more severe displacement preoperatively in the FPO group, the radiological results were similar at the end of the follow-up.List of scientific papersI. Kiapekos N, Broström E, Hägglund G, Åstrand P. Primary surgery to prevent hip dislocation in children with cerebral palsy in Sweden: a minimum 5-year follow-up by the national surveillance program (CPUP). Acta Orthop. 2019 Oct;90(5):495-500. https://doi.org/10.1080/17453674.2019.1627116II. Telléus A, Kiapekos N, Von Heideken J, Wagner P, Broström E, Hägglund G, Åstrand P. Orthopedic surgical procedures in 3,305 children and young adults with cerebral palsy: a register-based cohort study. Acta Orthop. 2022 May 23;93:472-477. https://doi.org/10.2340/17453674.2022.2583III. Kiapekos N, von Heideken J, Broström E, Hägglund G, Åstrand P. Treatment of Hip Displacement in Children With Cerebral Palsy: A 5-year Comparison of Proximal Femoral Osteotomy and Combined Femoral-Pelvic Osteotomy in 163 Children. J Pediatr Orthop. 2024 Jul 1;44(6):e536-e541. https://doi.org/10.1097/BPO.0000000000002674IV. Kiapekos N, Broström E, Hägglund G, Åstrand P. Radiographical development after Hip osteotomy in Children with Cerebral Palsy: A Five-Year Population-Based Study. [Manuscript]</p
Strategies for optimizing immune checkpoint inhibition in advanced non-small cell lung cancer
No full textAs the incidence of lung cancer surges, transcending traditional risk factors such as smoking, the medical community faces unprecedented challenges. Non-small cell lung cancer (NSCLC) diagnoses have risen across diverse demographics, including non-smokers, reflecting a shift in the disease's epidemiology. This thesis critically assesses the transformative role of immune checkpoint inhibitors (ICIs), a therapeutic class that has revolutionized the treatment of advanced NSCLC. While ICIs have improved survival rates, challenges remain, particularly unpredictable patient responses and maintaining quality of life. This work refines ICI treatment strategies to make them more personalized and effective, thereby shaping the future of cancer therapies and enhancing global patient outcomes.Paper I explores the corticosteroid use in 196 advanced NSCLC patients treated with ICIs at Karolinska University Hospital from 2016 to 2019. Patients were categorized based on steroid use for general support, palliative care, or immune- related adverse events (irAEs). Findings show that steroids for irAEs do not compromise survival (p = 0.38) and may serve as predictive markers, while those for palliative care are linked with significantly reduced survival (hazard ratio: 2.7; 95% confidence interval [CI], 1.5-4.9). These results underscore the importance of considering the rationale behind steroid use as a key prognostic and predictive factor. Given that corticosteroid use is a pressing clinical matter, seen daily in oncology practice, understanding its impact on patient outcomes is crucial for guiding treatment decisions in advanced NSCLC.Paper Il examines ICIs' intracranial effectiveness in 280 advanced NSCLC patients with brain metastases (BM), a group often excluded from trials. Despite neurological symptoms, ICIs achieved intracranial response rates of 24.2%, highlighting their potential in this underrepresented population. This study addresses a major gap in clinical research and advocates for the inclusion of advanced NSCLC patients with symptomatic BMs in future trials.Paper III identifies clinical factors-younger age (p = 0.024), previous radiation treatment (p = 0.027), and BM at diagnosis (p = 0.023)-that significantly improve intracranial disease control. These variables led to the development of predictive groups for disease progression (odds ratio: 27.5; p = 0.004). This research highlights the importance of clinical factors in determining intracranial effectiveness and calls for the development of robust biomarkers to guide ICI treatment decision.Paper IV introduces the Brain-Lung Immunotherapy Prognostic (BLIP) score, a novel tool for predicting outcomes in NSCLC patients with BMs undergoing ICI therapy. The BLIP score, validated in 152 patients, stratifies patients into "Good" and "Poor" prognostic groups with median intracranial survival of 15 months (95% CI: 2-4) and 7 months (95% CI: 4-10), respectively. Internal validation showed an AUC of 0.869 with external validation confirming its predictive power (p = 0.0063). This tool offers clinicians a reliable method to optimize ICI therapy and improve patient outcomes in a personalized manner.In conclusion, this thesis highlights the nuanced role of corticosteroids, the effectiveness of ICIs against BMs, and the importance of clinical factors guiding treatment. These findings advance our understanding of ICI therapy and advocate for more personalized treatment approaches that consider individual patient profiles. The implications of this research are twofold: it paves the way for more inclusive clinical trials and emphasizes the ongoing need for biomarkers to predict treatment outcomes. Ultimately, this thesis redefines treatment paradigms and sets new standards in oncology, inspiring future trials and potentially influencing global protocols to improve survival and quality of life for millions of NSCLC patients worldwide.List of scientific papersI. Skribek M, Rounis K, Afshar S, Grundberg O, Friesland S, Tsakonas G, Ekman S, De Petris L. Effect of corticosteroids on the outcome of patients with advanced non-small cell lung cancer treated with immune-checkpoint inhibitors. Eur J Cancer. 2021 Mar;145:245-254. Epub 2021 Jan 5. https://doi.org/10.1016/j.ejca.2020.12.012II. Skribek M, Rounis K, Makrakis D, Agelaki S, Mavroudis D, De Petris L, Ekman S, Tsakonas G. Outcome of Patients with NSCLC and Brain Metastases Treated with Immune Checkpoint Inhibitors in a 'Real- Life' Setting. Cancers (Basel). 2020 Dec 10;12(12):3707. https://doi.org/10.3390/cancers12123707III. Rounis K, Skribek M, Makrakis D, De Petris L, Agelaki S, Ekman S, Tsakonas G. Correlation of Clinical Parameters with Intracranial Outcome in Non-Small Cell Lung Cancer Patients with Brain Metastases Treated with Pd-1/Pd-L1 Inhibitors as Monotherapy. Cancers (Basel). 2021 Mar 29;13(7):1562. https://doi.org/10.3390/cancers13071562IV. Skribek M, Livanou M, Vathiotis I, Strandman V, Thorell A, Koulouris A, Syrigos K, Ekman S, Tsakonas G. The Validated Brain-Lung Immunotherapy Prognostic (BLIP) Score: A Novel Robust Tool for Prognostication in Non-Small Cell Lung Cancer Patients with Brain Metastases. [Manuscript]</p
Modulation of protective immunity in human tuberculosis
No full textThis thesis presents four studies aimed at elucidating immune responses to Mycobacterium tuberculosis (Mtb) infection, with a focus on the associations with clinical features, such as tuberculosis (TB) disease severity and anemia, and the role of histone deacetylase inhibitors (HDACi) in modulating these responses.In Study I, we developed a protocol for differentiation and polarization of monocyte-derived macrophages into M1 and M2 cells. Setting up a macrophage infection model with the virulent Mtb-H37Rv strain expressing green fluorescent protein (GFP), allowed assessment of the phenotype and function of Mtb-infected macrophages using flow cytometry. Results from this study indicated that Mtb modulates the polarization of macrophages and is efficiently internalized inside M2 polarized macrophages, which have lower capabilities of clearing the infection effectively. M1 macrophages, known for their robust response to bacterial infections, showed an overall lower Mtb-GFP signal potentially indicating an enhanced capacity for bacterial clearance, although limitations in bacterial containment were also noted in the M1 subset.In Study II, we investigated how peripheral inflammatory mediator and cytokine levels associated with distinct features in patients with pulmonary TB, including clinical disease severity and anemia. This TB patient cohort represented a sub- group obtained from a randomized clinical trial previously conducted in Ethiopia. Multiplex Magpix data from plasma was examined with machine learning and manual analyses which identified key inflammatory markers that differentiated between severe and mild TB, anemic and non-anemic cases, and patients with extensive and limited lung involvement. These analyses revealed effective ranking of immune markers that separated the immune responses at baseline and after the start of anti-TB treatment in the different patient groups and compared to the healthy controls. Notably, while certain markers correlated with immune protection, other markers decreased with treatment, highlighting their potential as biomarkers for disease progression and response to therapy.Previous work on the HDACi, phenylbutyrate (PBA), in the presence or absence of vitamin D, have demonstrated enhanced immune functions in Mtb-infected macrophages in vitro and clinical improvement of pulmonary TB upon administration to patients in vivo. However, PBA is a broad-spectrum HDACi with relatively weak HDACi activity in the millimolar-range. Therefore, Study III was designed to test a panel of commercially available HDACi compounds for their efficacy to reduce intracellular Mtb growth in immune cells. For this purpose, we used the macrophage infection model of Study I and IncuCyte live-cell imaging to enable medium-throughput screening of different compounds at different doses in cells obtained from healthy blood donors. The results demonstrated that four different SIRT1 and SIRT2 inhibitors, particularly tenovin, effectively enhanced restriction of Mtb growth in both Mtb-infected macrophages and bulk peripheral blood mononuclear cells (PBMCs). Specific SIRT2 inhibitors emerged as promising compounds useful for boosting intracellular bacterial clearance, although the efficacy of these molecules seemed dependent on the concentration and kinetics used. This study contributes to the growing body of research on HDACi as potential host-directed therapies for TB.To continue the exploration of immune responses in the clinical trial cohort used in Study II, the work in Study IV was focused on phenotypical and transcriptional profiling of peripheral immune cells in TB patients using bulk RNA-sequencing (seq) on FACS-sorted lymphocytes and myeloid cells in combination with flow cytometry. Bulk RNA-seq data obtained from patients with mild, moderate or severe TB disease as compared to the controls, revealed that several genes where differentially regulated in the CD4+, CD8+, and monocyte cell subsets. Moreover, markers such as CD25, CD38, and HLA-DR were elevated in TB patients, particularly in those with severe disease and anemia, and were linked to disease severity and immune activation. Activated T cells expressing Ox40 as well as CD25+FoxP3+ and CD38+HLA-DR+ T cells were associated with disease severity and of potential relevance in chronic TB. These markers and T cell subsets decreased with effective treatment, suggesting potential use as prognostic markers of treatment response.Together, these studies highlight significant insights into protective and pathological immune responses involved in TB, the impact of clinical features on immune responses, and the potential of HDAC inhibitors as adjunct host-directed therapies for modulation of TB immunity and to improve Mtb control.List of scientific papersI. Mily A, Kalsum S, Loreti MG, Rekha RS, Muvva JR, Lourda M, Brighenti S. Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon M. tuberculosis Infection. Journal of Visualized Experiments, 2020 Sep 18;(163). https://doi.org/10.3791/61807II. Ashenafi At, Loreti MG+, Bekele A, Aseffa G, Amogne W, Kassa E, Aderaye G, Brighenti S (+ Equal contribution) Inflammatory immune profiles associated with disease severity in pulmonary TB patients with moderate to severe clinical TB or anemia Frontiers in Immunology, 2023 Dec 12 (Vol 14 - 2023). https://doi.org/10.3389/fimmu.2023.1296501III. Kalsum S, Akber A, Loreti MG, Andersson B, Danielson E, Lerm M, Brighenti Sirtuin inhibitors reduce intracellular growth of M. tuberculosis in human macrophages via modulation of host cell immunity. [Submitted]IV. Loreti MG, Mily A, Akber M, Ashenafi S, Bekele A, Bekele A, Aseffa G, Amogne W, Kassa E, Aderaye G, Lourda M, Brighenti S Immune profiling of peripheral T cells and myeloid cells in patients with pulmonary TB exhibiting diverse disease severity traits. [Manuscript]</p
Superior vena cava blood flow in normal and growth restricted fetuses
No full textAssessing fetal well-being is crucial to enhance diagnosis, evaluate disease severity, optimize interventions when necessary, and improve perinatal outcomes. Doppler ultrasonography is widely used for noninvasive fetal surveillance, providing valuable insights into fetal physiology and hemodynamic changes in compromised fetuses. Intrauterine hypoxia initiates fetal adaptive hemodynamic responses, leading to increased blood supply to the brain and other vital organs at the expense of the placenta and lower body, a phenomenon known as "brain sparing". Currently, umbilical artery (UA) and middle cerebral artery (MCA) Doppler velocimetry, particularly the ratio of MCA pulsatility index (PI) and UA PI, is frequently employed to identify "brain sparing". However, it is important to note that the fetal response to hypoxia can impact both arterial circulation and venous return. We hypothesized that the quantification of SVC and umbilical vein (UV) blood flow could better reflect the circulatory balance between fetal cerebral and placental circulation.The purpose of this thesis was to study fetal brain circulation in the second half of pregnancy using Doppler ultrasonography with a focus on cerebral venous return through superior vena cava (SVC) in normal and growth restricted fetuses with the anticipation that it may lead to the development of a new surveillance method for high-risk pregnancies.Studies I-III were conducted on a low-risk pregnant population prospectively recruited at the Department of Obstetrics and Gynecology, University Hospital of North Norway, Tromsø, Norway, from 2009 to 2012. These studies were part of a longitudinal research project evaluating fetal hemodynamics in the second half of pregnancy. A total of 142 uncomplicated singleton pregnancies between 20-40 weeks' gestation were included, and the participants underwent ultrasound examinations at approximately 4-weekly intervals. Fetal weight was estimated using biometry of the head, abdomen, and femur. Doppler velocity waveforms of SVC, MCA, UA, and UV were recorded and analyzed offline. The diameters of SVC and UV were measured using two-dimensional ultrasound images. Volume blood flow (Q) of the SVC and UV were quantified. The conventional pulsatility index (PI)-based cerebroplacental (CPR) and umbilicocerebral (UCR) ratios and Q-based indices of brain sparing, Q-CPR, and Q-UCR were calculated.In Study I, we longitudinally investigated the ratio between cerebral and placental blood flow in the second half of normal pregnancies, quantifying volume blood flow in the SVC and UV during the second half of pregnancy. We established gestational age-specific reference intervals for Q-CPR and Q-UCR during the second half of pregnancy and found that there is a poor correlation and lack of agreement between PI-based and Q-based CPR and UCR. These results indicate that different physiological information may be obtained from venous and arterial circulation. Further investigation is necessary to see if these reference intervals are useful in fetal surveillance.In Study II, we established longitudinal reference values for fetal SVC blood velocities and pulsatility index for veins (PIV) in the second half of pregnancy. An automated tracing of SVC waveforms using the software of ultrasound machine provided the peak systolic velocity (PSV), peak diastolic velocity (D), time-averaged maximum velocity (TAMAXV), time-averaged mean velocity (TAMEANV), and end-diastolic velocity during atrial contraction (A-velocity). The pulsatility index for veins (PIV) was calculated. Our findings showed that SVC velocities increased as pregnancy advanced, while PIV did not change significantly. Normative values of SVC velocities and PIV could be useful for fetal hemodynamic assessment in high-risk pregnancies.In Study III, we aimed to quantify the proportion of fetal cardiac output (CO) distributed to the fetal brain and upper body during the second half of pregnancy using Doppler ultrasonography. To achieve this, we recorded the right and left cardiac ventricular outflow tract diameters and blood velocities and calculated the left, right, and combined cardiac output (CCO). We also estimated the SVC mean velocity from its TAMAXV and measured the SVC diameter to calculate the SVC volume blood flow (Q-SVC) using a mathematical equation, i.e., Q = mean velocity * cross-sectional area of the vessel. Q-SVC normalized for estimated fetal weight (Q-SVCw) was also calculated. The results showed that 10% of the SVC fraction of fetal CCO, calculated as (Q-SVC/CCO) * 100, was distributed to the brachio-cephalic circulation. This fraction remained constant during the latter half of pregnancy.In study IV, we extended our research on SVC Doppler to pregnancies complicated by reduced fetal growth. We included 40 singleton pregnancies examined between 32+0 to 36+6 weeks of gestational age with estimated fetal weight (EFW)In summary, we established normal reference ranges for fetal SVC volume blood flow, Doppler velocities, and PIV. We developed new volume blood flow-based indices of fetal brain sparing, constructed reference charts and demonstrated their usefulness in studying cerebral hemodynamic alterations in fetuses with late onset growth restriction. The findings of our studies may potentially have a wider application in future research and clinical practice.List of scientific papersI. Volume blood flow-based indices of fetal brain sparing in the second half of pregnancy: A longitudinal study. Stefopoulou M, Johnson J, Wilsgaard T, Lindgren P, Herling L, Kiserud T, Acharya G. Acta Obstet Gynecol Scand. 2020 Dec;99(12):1717-1727. Epub 2020 Jul 16. https://doi.org/10.1111/aogs.13950II. Reference ranges of fetal superior vena cava blood flow velocities and pulsatility index in the second half of pregnancy: a longitudinal study. Stefopoulou M, Herling L, Johnson J, Lindgren P, Kiserud T, Acharya G. BMC Pregnancy Childbirth. 2021 Feb 23;21(1):158. PMCID: PMC7901110https://doi.org/10.1186/s12884-021-03635-6III. Fetal superior vena cava blood flow and its fraction of cardiac output: A longitudinal ultrasound study in the second half of pregnancy. Stefopoulou M, Johnson J, Herling L, Lindgren P, Kiserud T, Acharya G. Front Pediatr. 2021 Jul 6;9:658502. PMCID: PMC8289903https://doi.org/10.3389/fped.2021.658502IV. Fetal superior vena cava blood flow and volume blood flow-based indices of brain sparing in fetuses with an estimated weight below the 10th percentile during the third trimester of pregnancy. Stefopoulou M, Johnson J, Lindgren P, Acharya G [Manuscript]</p