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Evaluation of new and current preventive strategies for cervical and vaginal cancer
No full textThis thesis addresses clinical research questions on primary and secondary prevention of cervical and vaginal cancer by targeting four groups of women in the organized cervical screening program; women entering the screening program, women exiting the screening program, non-attenders in the screening program, and women previously treated for precancerous lesions.In Study 1, we evaluated the colposcopic and histopathologic findings in women with 1 and 3 year human papillomavirus (HPV) persistence, respectively, in women exiting the cervical screening program at age 56-64. The design was a randomized health care policy offering HPV screening to 50% of resident women exiting the screening program in the Stockholm-Gotland region of Sweden during January 2012 through May 2014. Women with HPV persistence were referred to colposcopy. Biopsies as well as an endocervical sample analyzed for cytology and HPV was taken at the examination. HPV was analyzed for 14 different possibly oncogenic genotypes. We found that HPV prevalence was 5.5% (405 women of 7325 attending). After one year, 91 of 176 (52%) were persistently HPV positive and after three years 55 of 137 (40%) (p=0.042). Histopathologically confirmed cervical intraepithelial lesions grade 2 or worse (CIN2+) was found in 19 of 82 women (23%) and 9 of 45 women (20%) in the 1 and 3 year groups, respectively, indicating that it was safe to postpone repeat HPV testing for three years in women exiting the cervical screening program. There was a high risk for CIN2+ at follow-up and noteworthy yields from HPV genotyping as well as endocervical sampling and random biopsies in the absence of visible colposcopic lesions.In Study 2, we compared the colposcopic evaluation in vaccinated and unvaccinated women entering the organized cervical screening program. Women in the 1994 and 1995 birth cohorts who entered the cervical screening program at age 23 in one region in Sweden were identified. Colposcopy was performed within two to four months after a positive screening result and evaluated with two different tests; Swedescore and Colposcopic impression, respectively. Endocervical sampling with analysis for cytology and 14 possibly oncogenic HVP genotypes, as well as punch biopsies for histopathologic confirmation were taken in all women. Out of 165 women with positive screening tests, 160 (98%) attended colposcopy, of which 90 (56%) were vaccinated and 70 (44%) were unvaccinated. Only 7 out of 90 (5%) women in the vaccinated group were HPV 16/18-positive, compared with 23 out of 70 (33%) in the unvaccinated group (PIn Study 3, we identified all women aged 33–62 who had not participated in the organized cervical screening program for at least 10 years in one Swedish county. HPV self-sampling kits were sent to all eligible women. A randomized written reminder was sent to non-responders after 8 weeks. HPV-positive women were referred directly to colposcopy without prior triage. Biopsies for histopathologic confirmation were taken in all women. Among eligible women, 150/741 (20.2%) returned the self-sample kit or attended routine screening; 11 of the responders returned the kit after the reminder. In total, 23/150 (15.3%) of returned kits were HPV positive. Out of the 23 HPV-positive women, 17 (74%) attended colposcopy; 11/17 (65%) had a histopathological high-grade intraepithelial lesions (HSIL) or an invasive cervical cancer. The direct send kit strategy and referral of all HPV-positive women to colposcopy without prior triage appears to be feasible if resources are available and should be prioritized given the high prevalence of HSIL lesions and cancer among non- attenders.In Study 4, we identified women resident in Sweden between 1 Jan 1999 and 31 Dec 2018 and diagnosed with HSIL/adenocarcinoma in situ (AIS) at age 22 or above and their outcome of interest (cervical and vaginal cancer) through the National Cancer Register (NCR). Women were linked to the Swedish National Cervical Screening Registry (NKCx) to retrieve information on cervical screening history including cytology and HPV tests. After various exclusions and censoring for hysterectomy, there were 67693 women at baseline out of which 213 were identified as having a subsequent cervical or vaginal cancer. Testing was divided into four different states defined by test of cure vs no test of cure stratified by follow-up testing: 1) no follow-up, 2) irregular follow-up, 3) regular follow-up, and 4) the time period after being eligible for the test of cure and before initiating follow-up. For each state, we found an excess risk of invasive cervical or vaginal cancer for women without a test of cure: no follow- up (HR 13.9, CI 1.6-121.1), irregular follow-up (HR 2.3, CI 1.1-4.7), regular follow-up (HR 1.4, CI 0.8-2.2), the time period after being eligible for the test of cure and before initiating follow-up (HR 4.8, CI 1.8-13.1), though the excess was modest and statistically not significant for regular follow-up. The results indicate that efforts to increase participation in follow-up during the first three years after treatment of HSIL/AIS should be prioritized.List of scientific papersI. Sahlgren H, Elfström KM, Lamin H, Carlsten-Thor A, Eklund C, Dillner J, Elfgren K. Colposcopic and histopathologic evaluation of women with HPV persistence exiting an organized screening program. Am J Obstet Gynecol. 2020 Mar;222(3):253.e1-253.e8. https://doi.org/10.1016/j.ajog.2019.09.039 II. Sahlgren HAI, Elfgren K, Sparen P, Elfstrom MK. Colposcopic performance in a birth cohort previously eligible for human papillomavirus vaccination. Am J Obstet Gynecol. 2022 May;226(5):704.e1-704.e9. https://doi.org/10.1016/j.ajog.2021.11.1372 III. Sahlgren H, Sparén P, Elfgren K, Miriam Elfström K. Feasibility of sending a direct send HPV self-sampling kit to long-term non- attenders in an organized cervical screening program. Eur J Obstet Gynecol Reprod Biol. 2022 Jan;268:68-73. https://doi.org/10.1016/j.ejogrb.2021.11.430 IV. Milerad H, Sparen P, Johansson ALV, Elfgren K, Andrae B, Ploner A, Elfström M (joint last authorship). Screening behaviour after HSIL/AIS and the risk of invasive cervical and vaginal cancer: A nationwide cohort study 1999-2018. [Manuscript]</p
Vacuum extraction and pelvic floor injury : a randomized controlled trial and cohort studies
No full textBACKGROUND AND AIMS: Pelvic floor dysfunction (PFD) affects many women, especially after complicated childbirth. The impact of a prolonged second stage on PFD is not clear, while the effects of operative vaginal birth (OVB) and obstetric anal sphincter injury (OASI) are better known, especially concerning the risk of anal incontinence (AI). Vacuum extraction (VE) and primiparity are the major risk factors of OASI. Observational studies report that OASI in VE in nulliparous women decreases if an episiotomy is performed, but this has not been confirmed in a randomized controlled trial (RCT). The aim of this thesis was to investigate factors that could improve second stage management, particularly in VE, to decrease the risk of OASI and future PFD. First, we aimed to identify risk factors for OASI in VE. Second, we aimed to explore effects of a prolonged second stage on PFD, and finally, we aimed to investigate if a lateral episiotomy, compared with no episiotomy, reduces the risk of OASI in VE in nulliparous women.METHODS AND MAIN RESULTS: Study I is a retrospective cohort study with one-year medical records data from Danderyd Hospital. All primiparous women with a live, singleton fetus ≥34 gestational weeks, delivered by VE during 2013 were included (n=323). Primary outcome was OASI and exposure was operator category (obstetrician, gynecologist, and resident) with obstetrician as reference. OASI occurred in 57 (17.6%) women. Fifteen (11.5%) OASI occurred in VE performed by obstetricians, 10 (13.5%) by gynecologists (aOR 1.84, 95% CI 0.72-4.70), and 32 (26.9%) by residents (aOR 5.13, 95% CI 2.20-11.95). Study II and III are questionnaire and cohort studies including primiparous women with a live, single fetus in cephalic presentation ≥37 gestational weeks and second stage duration ≥3 h in the Stockholm Region during one year (2019, n=1302). Data were retrieved from electronic medical records (EMR). The oneyear follow-up questionnaire from the Swedish Perineal Laceration Register, including questions regarding symptoms of urinary incontinence (UI), anal incontinence (AI), and pelvic organ prolapse (POP) were distributed one to two years after delivery. In Study II, primary outcome was AI defined as Wexner score ≥2. Main exposure was mode of delivery with cesarean section (CS) as reference. Secondary exposures were degree of perineal injury and extended second stage duration. We found that the odds of AI were increased by VE (aOR 2.25, 95% CI 1.21-4.18) but not by spontaneous vaginal birth (SVB) (aOR 1.55, 95% CI 0.85-2.84). AI was also increased by OASI (aOR 2.03, 95% CI 1.17-3.52) and second-degree perineal injuries (aOR 1.36, 95% CI 1.03-1.81). OASI and VE combined inferred the highest odds (aOR 4.06, 95% CI 1.80-9.14) compared with CS. Extended duration of the prolonged second stage did not affect the risk of AI. In Study III, primary outcome was a composite of PFD including at least weekly symptoms of UI, AI, and POP. Exposure was intervention with VE or CS at 3-4 h or at 4-5 h respectively, compared with expectant management. The risk of PFD was increased after VE at 3-4 h (aRR 1.33, 95% CI 1.06-1.65) and 4-5 h (aRR 1.34, 95% CI 1.05-1.70), but remained unchanged after CS. The increased risk after VE was not mediated by OASI. Study IV is a multicenter RCT of lateral episiotomy compared with no episiotomy in nulliparous women with a live, single, fetus ≥34 gestational weeks, requiring VE during 2017-2023. The intervention was a lateral episiotomy. Primary outcome was OASI. The modified intention-to-treat (mITT) population included women with attempted or successful VE (n=702). In the intervention group, 21/344 (6.1%) women sustained OASI compared with 47/358 (13.1%) in the comparison group (p=0.002). The risk difference was -7.0% (96% CI -11.7% to - 2.5%). The unadjusted risk ratio was 0.46 (96% CI 0.28-0.78) and 0.47 (96% CI 0.23-0.97) adjusted for site. Number needed to treat was 14.3 to avoid one OASI. Wound infection and dehiscence were significantly increased in the intervention group, while all other outcomes were similar.CONCLUSION: In VE in nulliparous women, the risk of OASI increased when the operator was a resident, indicating a need for increased training and supervision. In a prolonged second stage, an extended duration did not increase the risk of AI or PFD. If a SVB seems likely, it is better to wait than to intervene with VE in order to avoid future PFD. If intervention is necessary, and several risk factors for OASI are present, CS could be preferable to reduce the risk of AI. In VE in nulliparous women, a lateral episiotomy significantly reduces the risk of OASI. However, the intervention may increase the risk of wound infection and dehiscence.List of scientific papersI. Operator experience affects the risk of obstetric anal sphincter injury in vacuum extraction deliveries. Bergendahl S, Lindberg P, Brismar Wendel S. Acta Obstetricia et Gynecologica Scandinavica. 2019 Jun;98(6):787-794.https://doi.org/10.1111/aogs.13538II. Anal incontinence after a prolonged second stage of labor in primiparous women. Bergendahl S, Sandström A, Spasojevic A, Brismar Wendel S. Scientific reports. 2022 May;12(1):7315.https://doi.org/10.1038/s41598-022-11346-xIII. Pelvic floor dysfunction after intervention compared to expectant management in prolonged second stage of labour: A population-based questionnaire and cohort study. Bergendahl S, Sandström A, Zhao H, Snowden J, Brismar Wendel S. https://doi.org/10.1111/1471-0528.17792IV. Lateral episiotomy or no episiotomy in vacuum assisted delivery in nulliparous women: a randomized, open-label, superiority trial. Bergendahl S, Jonsson M, Hesselman S, Ankarcrona V, Leijonhufvud Å, Wihlbäck A-C, Wallström T, Rydström E, Friberg H, Kopp Kallner H, Brismar Wendel S. [Submitted]</p
Patient choice and provider incentives : socioeconomic differentials in effects from market reform in Swedish primary care
No full textDuring the last decades, market reforms have been increasingly implemented in publicly financed health care systems. Still, there is a lack of knowledge of how these reforms affect inequality in access to care and in turn health inequality. This thesis aims to provide such evidence.The overarching aim of the thesis is to increase the understanding of how market mechanisms affect the socioeconomic (SES) distribution of access to, utilization of, and outcomes from, primary health care (PHC) in a publicly financed health care system. The thesis also aims to further disentangle the effect of three interacting market mechanisms: patient choice, provider location, and changes in reimbursement models. We operationalize our aim using a set of reforms implemented in Swedish PHC in 2007-2009.The thesis consists of four sub-studies which are all based on comprehensive individual level total population registry data. Study I explores SES differentials in PHC utilization, using concentration indices and data for the three largest Swedish regions, which implemented the reforms at slightly different points in time. Study II estimates the causal separate but interdependent effects of PHC unit location and patient choice on SES differences in patient mix across PHC units, using data from two regions, a counterfactual approach and a decomposable segregation index. Study III assesses to what extent reform exposure had differential impact on PHC outcome quality for low-income elders as compared to matched peers, using a difference-in-difference-in-differences approach, comparing the development in one region exposed to reform, to that of another (at the time) unexposed region. Outcome quality is approximated using avoidable hospitalizations. Finally, Study IV exploits the fact that a smaller set of PHC units in one region for a transitional period did not enter into a new reimbursement design that included part per visit payments. We leverage this variation using difference-in-differences models, and compare the development of PHC visits, secondary care outpatient visits and avoidable hospitalizations by age group, chronic disease and SES factors.We find that following market reform, individuals with lower SES increased their number of visits more than those with higher SES. This occurred to some extent across all studied regions, but was more pronounced and clearly reform-timed in the region that implemented part per visit payments. Further, within the region with part per visit payments, the effect was slightly larger for PHC units exposed to the new reimbursement design, compared to a subset of units that temporarily remained unexposed. In the latter case, differences were however modest and specific to older individuals. The higher increase in the number of visits for individuals with lower SES was partly explained by visits close in time to a previous visit, and particularly so in the presence of part per visit payments. This may reflect a subdivision of visits in response to reimbursement incentives particularly for individuals with lower SES, but since data do not reveal the relevance, content, length or quality of each visit, the exact interpretation of the increases in recurrent visits remains to be further understood. We also find indications that PHC visits may have substituted secondary outpatient care when PHC units were in part paid per visit, but these findings, and their qualitative implications for individuals with different SES, also need to be further explored.We further find that market reform increased differences in SES patient mix between PHC units. This was mainly due to a smaller group of new PHC units in the capitation-based region locating in low SES areas, rather than PHC units selecting high SES patients or patients clustering to certain providers through choice patterns. It remains to be explored if the geographical access increases in lower SES areas translated into increased utilization of PHC or, for that matter, better health outcomes.Finally, we find that market reform had on average negative effects on health care quality for individuals with lower SES, when measured as avoidable hospitalizations in a specific population of older adults. Results are ambiguous as to whether this differed by reimbursement model.The thesis takes a comprehensive scope on SES differentials in effects from market reform in publicly financed PHC. It thereby contributes with both broad and in-depth empirical evidence to a sparsely researched but highly policy relevant research area. The findings suggest that market reforms do influence socioeconomic differentials in PHC, and that reimbursement models may play an important role in shaping these effects. It concludes that, for individuals with lower SES, in the largest Swedish regions, market reforms appear to have increased health care access, both in terms of geographical proximity and visit volume, but effects on health care content and quality are either unknown, ambiguous or point to negative effects that are specific to individuals with lower SES.The results call for vigilance and curiosity regarding potential heterogeneities in effects of market models in PHC. Most importantly, they emphasize that policymakers need to closely and continuously monitor provider reimbursement models, as their effects - though potentially modest on average - may disproportionately impact individuals with lower SES. The findings also highlight the need for future studies to employ more granular data on diagnoses and treatments to better understand how market reforms impact the content and quality of care.List of scientific papersI. Sveréus S, Kjellsson G, Rehnberg C. Socioeconomic distribution of GP visits following patient choice reform and differences in reimbursement models: Evidence from Sweden. Health Policy. 2018 Sep;122(9):949-956. https://doi.org/10.1016/j.healthpol.2018.07.017II. Sveréus S, Kjellsson G, Rehnberg C. Market reform and socio-economic segregation in primary care - a counterfactual approach to separating the effects of provider location and patient choice. [Manuscript]III. Sveréus S, Petzold M, Rehnberg C. Change in avoidable hospitalizations for low-income elders following quasi-market reform in primary care - Evidence from a natural experiment in Sweden. Soc Sci Med. 2024 Apr;346:116711. https://doi.org/10.1016/j.socscimed.2024.116711IV. Sveréus S, Rehnberg C, Kjellsson G. Heterogeneous effects of part per visit payments in primary care: insights from a natural experiment in Sweden. [Manuscript]</p
Treatment outcomes following alveolar cleft rehabilitation
No full textIntroduction: Alveolar cleft closure is typically done with bone grafting, and bone healing is assessed radiologically and clinically. However, there is no consensus on the best radiological follow-up protocol, and opinions vary regarding the periodontal health of teeth in the cleft region. Additionally, patients' self- assessment of treatment outcomes, though crucial, is sometimes underemphasized.Aims: The aim of this project was to investigate the treatment outcomes following alveolar cleft rehabilitation, focusing on the clinical periodontal status and radiological bone support for the teeth in the cleft region. Additionally, the project aimed to determine if the radiation dose in CBCT examinations could be reduced and to validate different methods for evaluating bone healing after bone grafting in the cleft region using CBCT images. Another aim was to assess the subjective treatment outcomes in terms of self-perceived OHRQoL after being treated according to a multidisciplinary cleft program.Material and methods: The thesis includes four studies comprising between 23 and 72 patients born with cleft lip, and alveolus (CLA) or cleft lip, alveolus, and palate (CLP). In a cross-sectional study, the clinical periodontal status and radiological bone support for the teeth in the cleft region were examined at 19 years of age (almost a decade after bone grafting the alveolar cleft) in patients with unilateral cleft lip and palate (UCLP). Bone support for the teeth was assessed using the Bergland Index (BI), which measures vertical bone height on intra-oral radiographs. To evaluate clinical periodontal status, periodontal probing depths and the presence of gingival recessions were measured, along with the presence of gingival inflammation. A total of 40 patients were examined clinically and 39 radiologically. Gingival health in the cleft region was compared with the corresponding teeth on the non-cleft side in a split-mouth design.To explore the possibility of reducing radiation doses in CBCT examinations for evaluating the alveolar cleft before or after bone grafting, two different radiographic protocols were compared in terms of image quality. In this randomized clinical trial (RCT), patients with CLA or CLP at an average age of 9.5 years were randomized for CBCT examination of the alveolar cleft to either an SD or ULD protocol, with 36 patients in each group. To assess image quality, the reviewers evaluated how well they could see the anatomical structures of interest. Image quality was graded on a three-level scale.CBCT scans taken an average of 6.6 months after bone grafting the alveolar cleft in 23 patients born with CLA or CLP were reviewed to validate and compare different methods for assessing the outcome of bone grafting using CBCT. The average age at the time of bone grafting was nine years. Volumetric bone fill (BF) was calculated, and bone healing was assessed using the Suomalainen and Liu grading scales. The outcome of the bone grafting was based on medical records from a multidisciplinary expert consensus meeting, classified as success or regraft. The outcome of the bone grafting was compared with volumetric BF and the results from the Suomalainen and Liu grading scales. Reliability for the different variables was analyzed using intra-class correlation and by calculating the kappa-value.To assess the subjective treatment outcome at 19 years of age, OHRQoL was examined using questionnaires. The questionnaires, including the Oral Health Impact Profile-14 (OHIP-14), Jaw Functional Limitation Scale-20 (JFLS-20), and Orofacial Esthetic Scale (OES), were completed at the last follow-up meeting by 32 patients born with UCLP. The results were compared with two age-matched groups born without cleft: one group that had undergone orthodontic treatment and one that had not.Results: Bergland Index (BI) I and II were observed in 87% of the patients. Few pathological pockets were recorded, and there was no statistically significant difference in periodontal pocket depth on the cleft side compared to the corresponding sites on the non-cleft side. There was a statistically significant higher percentage of examined sites with gingival recession on the cleft side (6.6%) compared to the non-cleft side (1.7%). Additionally, the gingival index was significantly higher on the cleft side than on the non-cleft side.When comparing the image quality of the different protocols for CBCT examinations in the cleft region, ULD and SD protocols, no statistically significant differences were found in structure visibility regarding anatomical structures of interest.The validation of different methods for quantifying bone healing after bone grafting to the cleft region showed that the calculation of volumetric BF and the Suomalainen grading scale had high validity, while the Liu grading scale showed low validity. All methods had high reliability.In the evaluation of OHRQoL, there was no significant difference in the mean summary scores on the JFLS-20 and OHIP-14 between patients born with cleft and the two groups born without cleft. The orthodontically treated group born without cleft had a significantly higher OES mean summary score than those born with UCLP.Conclusions: At 19 years of age, the teeth within the bone-grafted cleft region typically have good bone support according to the BI. The teeth in the cleft region exhibit a higher degree of gingival inflammation compared to the non- cleft side, which may eventually lead to periodontitis, tooth loss, and other complications affecting overall health and well being.For the examination of the alveolar cleft with CBCT, the ULD protocol is recommended over the SD since the radiation dose is lower and the diagnostic information obtained about the cleft region is equivalent.For follow-up and quantification of bone healing with CBCT, volumetric 3D calculation and the Suomalainen grading scale are recommended, and a pre- operative CBCT is not required. However, an individual assessment must always be made for each patient to determine the need for regrafting.Self-perceived OHRQoL in the studied population is generally similar between patients who have undergone treatment for UCLP and their peers, with the UCLP group perceiving their orofacial appearance similarly to those who have not had orthodontic treatment. However, individuals who have undergone orthodontic treatment tend to perceive their orofacial appearance more positively.List of scientific papersI. Long-term radiographic and periodontal evaluations of the bone- grafted alveolar cleft region in young adults born with a UCLP. Lemberger M, Peterson P, Andlin-Sobocki A, Setayesh H, Karsten A. European Journal of Orthodontics 2024; 46 (1). https://doi.org/10.1093/ejo/cjad064II. Low-dose cone-beam computed tomography for assessment of alveolar clefts: A randomized controlled trial in image quality. Lemberger M, Regnstrand T, Karsten A, Benchimol D, Shi XQ. Plastic Reconstructive Surgery 2024; 153 (4): 897-903. https://doi.org/10.1097/PRS.0000000000010588III. Validation and comparison of 2D grading scales and 3D volumetric measurements for outcome assessment of bone-grafted alveolar clefts in children. Lemberger M, Benchimol D, Pegelow M, Jacobs R, Karsten A. European Journal of Orthodontics. 2024; 46(2). https://doi.org/10.1093/ejo/cjae002IV. Oral health related quality of life following multidisciplinary treatment in young adults born with unilateral cleft lip and palate. Lemberger M, Pegelow M, Peterson P, Larsson P, Karsten A. [Manuscript]</p
Topoisomerase regulation in the interplay between DNA transcription and replication
No full textDuring DNA-involving processes such as transcription and replication the double helix is subjected to torsional stress. The over- or under-winding that encompasses torsional stress can be resolved by enzymes called topoisomerases. These enzymes cleave either one (Topoisomerase 1) or both (Topoisomerase 2) of the DNA strands, which allows for DNA relaxation, before resealing the break. Topoisomerase activity can be regulated by transcription or replication factors to fine-tune the efficiency of the genomic processes in which they are involved. Because cancers cells heavily rely on topoisomerases, these enzymes are common targets in cancer treatment.In this thesis, I have investigated topoisomerase regulation during transcription and replication, as well as new pharmacological strategies targeting the regulation of topoisomerase activity. The long-term goal of this thesis project is to develop approaches to selectively affect cancer cells viability. In paper 1, we focused on studying how the RNA polymerase 2 modulates Topoisomerase 1 during mitotic transcription. In paper 2, we investigated the mechanism of cellular adaptation to Topoisomerase 1 inhibitors. In paper 3, we developed a therapeutic approach that blocks the interaction between RNA polymerase 2 and Topoisomerase 1. We tested the strategy in preclinical models of pancreatic cancers. We showed that the treatment is effective and specific for the cancer cells and we have characterized the mechanism of cell killing.The thesis work integrates genomics, molecular biology, and drug screen methodologies, as well as preclinical mouse work done in collaboration. This works identifies Topoisomerase 1 as an important player in ensuring correct recruitment of various factors needed for transcription elongation, splicing and termination. The findings increase our understanding of regulatory pathways controlled by Topoisomerase 1 to protect DNA integrity and provide the rationale for the development of new approaches that target Topoisomerase 1 for cancer treatment.List of scientific papersI. "Topoisomerase 1 activity during mitotic transcription favors the transition from mitosis to G1" - Anika Wiegard, Vladislav Kuzin, Donald P. Cameron, Jan Grosser, Michele Ceribelli, Rashid Mehmood, Roberto Ballarino, Francesco Valant, Radoslaw Grochowski, Ivana Karabogdan, Nicola Crosetto, Arne Lindqvist, Anna Helene Bizard, Fedor Kouzine, Toyoaki Natsume, and Laura Baranello; Molecular Cell 81, 5007-5024.https://doi.org/10.1016/j.molcel.2021.10.015II. "NEDDylated Cullin 3 mediates the adaptive response to topoisomerase 1 inhibitors" - Alice Meroni, Jan Grosser, Sumedha Agashe, Natasha Ramakrishnan, Jessica Jackson, Priyanka Verma, Laura Baranello, Alessandro Vindigni; Sci. Adv. 8, eabq0648 (2022).https://doi.org/10.1126/sciadv.abq0648III. "Coinhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills pancreatic cancer via readthrough transcription" - Donald P. Cameron*, Jan Grosser*, Swetlana Ladigan*, Vladislav Kuzin, Evanthia Iliopoulou, Anika Wiegard, Hajar Benredjem, Kathryn Jackson, Sven T. Liffers, Smiths Lueong, Phyllis F. Cheung, Deepak Vangala, Michael Pohl, Richard Viebahn, Christian Teschendorf, Heiner Wolters, Selami Usta, Keyi Geng, Claudia Kutter, Marie Arsenian-Henriksson, Jens T. Siveke, Andrea Tannapfel, Wolff Schmiegel, Stephan A. Hahn, Laura Baranello; Sci. Adv. 9, eadg5109 (2023).https://doi.org/10.1126/sciadv.adg5109</p
Molecular phenotyping of midbrain dopaminergic neurons derived from human induced pluripotent stem cells
No full textThe objective of this study is to investigate the generation, maturation, and function of midbrain dopaminergic (mDA) neurons derived from human induced- pluripotent stem cells (iPSCs), alongside examining the mechanisms underlying their degeneration in Parkinson's disease (PD). This research aims to advance cell replacement therapies, disease modeling, and the identification of potential therapeutic targets.To achieve this, we first developed a novel mDA neuron differentiation protocol using human embryonic stem cells (hESCs) that accurately recapitulates key aspects of in vivo human ventral midbrain development (Paper I). This approach employs a combination of dual WNT activation, CHIR boost and LN511 coating to improve midbrain patterning. In addition, neurogenesis and differentiation were further enhanced by activating liver X receptors and inhibiting fibroblast growth factor (FGF) signaling.We next leveraged the mDA neuron differentiation protocol as a blueprint for iterative refinements, aiming to adapt and optimize it for various human induced stem pluripotent cell (hiPSC) lines to ensure broad applicability. These efforts led to a streamlined differentiation protocol, which was validated across multiple iPSC lines (Paper II). Additionally, a time-course multiomic analysis of mDA neuron differentiation identified discrepancies in gene expression and regulation patterns compared to human midbrain development. This information was utilized to correct the initial protocol, optimizing regional specificity, cell type composition, molecular signatures, and maturation of mDA neurons, ultimately leading to enhanced recovery in PD animal models (Paper III).On the other hand, we explored phenotypic changes associated with PD, focusing on mutations in the GBA gene, which encodes the glucosylceramidase enzyme, by comparing GBA E326K and N370S mutated iPSCs to their isogenic controls. Through secretome profiling of cerebrospinal fluid (CSF) and iPSC- derived mDA neurons from PD patients, we determined mDA neuron-attributed alterations in CSF protein composition and found that FKBP4, a member of immunophilin protein family, has a potential linkage to pathological progression and psychiatric symptoms in GBA mutation-associated PD (Paper IV). Using both 2D monocultures and a microfluidic brain-on-a-chip model, we revealed that GBA E326K mutated mDA neurons exhibited accelerated differentiation and altered axonal morphology. These findings are consistent with molecular phenotypes identified through the single-nuclei RNA sequencing, which showed that mutated midbrain progenitors displayed expedited neurogenesis, while their descendant neurons progressed towards neurodegenerative phenotypes (Paper V).In summary, this study advances our understanding of mDA neuron biology and PD pathogenesis, providing a strong foundation for further research in regenerative medicine and therapeutic development.List of scientific papersI. Single-cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation. Kaneyasu Nishimura, Shanzheng Yang, Ka Wai Lee, Emilía Sif Ásgrímsdóttir, Kasra Nikouei, Wojciech Paslawski, Sabine Gnodde, Guochang Lyu, Lijuan Hu, Carmen Saltó, Per Svenningsson, Jens Hjerling-Leffler, Sten Linnarsson, Ernest Arenas. Stem Cell Reports. 2023. https://doi.org/10.1016/j.stemcr.2022.10.016II. A reference human induced pluripotent stem cell line for large-scale collaborative studies. Caroline B Pantazis, Andrian Yang, Erika Lara, Justin A McDonough, Cornelis Blauwendraat, Lirong Peng, Hideyuki Oguro, Jitendra Kanaujiya, Jizhong Zou, David Sebesta, Gretchen Pratt, Erin Cross, Jeffrey Blockwick, Philip Buxton, Lauren Kinner-Bibeau, Constance Medura, Christopher Tompkins, Stephen Hughes, Marianita Santiana, Faraz Faghri, Mike A Nalls, Daniel Vitale, Shannon Ballard, Yue A Qi, Daniel M Ramos, Kailyn M Anderson, Julia Stadler, Priyanka Narayan, Jason Papademetriou, Luke Reilly, Matthew P Nelson, Sanya Aggarwal, Leah U Rosen, Peter Kirwan, Venkat Pisupati, Steven L Coon, Sonja W Scholz, Theresa Priebe, Miriam Öttl, Jian Dong, Marieke Meijer, Lara J M Janssen, Vanessa S Lourenco, Rik van der Kant, Dennis Crusius, Dominik Paquet, Ana-Caroline Raulin, Guojun Bu, Aaron Held, Brian J Wainger, Rebecca M C Gabriele, Jackie M Casey, Selina Wray, Dad AbuBonsrah, Clare L Parish, Melinda S Beccari, Don W Cleveland, Emmy Li, Indigo V L Rose, Martin Kampmann, Carles Calatayud Aristoy, Patrik Verstreken, Laurin Heinrich, Max Y Chen, Birgitt Schüle, Dan Dou, Erika L F Holzbaur, Maria Clara Zanellati, Richa Basundra, Mohanish Deshmukh, Sarah Cohen, Richa Khanna, Malavika Raman, Zachary S Nevin, Madeline Matia, Jonas Van Lent, Vincent Timmerman, Bruce R Conklin, Katherine Johnson Chase, Ke Zhang, Salome Funes, Daryl A Bosco, Lena Erlebach, Marc Welzer, Deborah Kronenberg-Versteeg, Guochang Lyu, Ernest Arenas, Elena Coccia, Lily Sarrafha, Tim Ahfeldt, John C Marioni, William C Skarnes, Mark R Cookson, Michael E Ward, Florian T Merkle. Cell Stem Cell. 2022. https://doi.org/10.1016/j.stem.2022.11.004III. Single-cell multiomic sequencing informs strategies to enrich the differentiation of human iPS cells in SOX6+ midbrain dopaminergic neurons. Guochang Lyu, Anqi Xiong, Rika Kojima, Judith Kreutzmann, Wojciech Paslawski, Chiara Tremolanti, Carmen Salto, Per Uhlén, Per Svenningsson, Ernest Arenas. [Manuscript]IV. Secretome Analyses Identify FKBP4 as a GBA1-Associated Protein in CSF and iPS Cells from Parkinson's Disease Patients with GBA1 Mutations. Rika Kojima, Wojciech Paslawski, Guochang Lyu, Ernest Arenas, Xiaoqun Zhang, Per Svenningsson. International Journal of Molecular Sciences. 2024. https://doi.org/10.3390/ijms25010683V. A comprehensive phenotypical characterization of midbrain dopaminergic neurons derived from GBA E326K Parkinsonian iPSCs. Guochang Lyu, Rika Kojima, Anqi Xiong, Judith Kreutzmann, Wojciech Paslawski, Carmen Salto, Inês Sousa Pereira, Clelia Introna, Maria José Lopez Martinez, Josep Samitier Martí, Josep M. Canals Coll, Per Uhlén, Per Svenningsson, Ernest Arenas. [Manuscript]</p
Strategies for improving melanoma survival : from early detection to therapies for advanced disease
No full textCM is one of the deadliest skin cancers, with increasing incidence in a majority of countries with predominantly fair-skinned populations. Despite the introduction of systemic therapies for metastasized CM showing promising effects on survival, CM is still responsible for considerable morbidity and mortality.The aim of this thesis is therefore to investigate the potential role of AI as a tool to improve clinicians' performance in CM diagnosis, to study the effect of some commonly used drugs with a putative immune modulatory effect on CM survival, and to investigate the outcome of systemic therapies and clinical markers in patients with metastatic CM in a real-world setting.In study I, we conducted a systematic review and meta-analysis of use of AI- assistance for skin cancer diagnosis including CM. In our search, we identified 2'983 studies, of these, ten could be included for meta-analysis. The results indicated that clinicians within all levels of experience benefitted from AI-assistance and those with least experience in dermatology showed the most significant improvement. However, only few studies were conducted in real-life settings.In study II, we performed a population-based cohort study of patients diagnosed with a primary invasive CM in Sweden between 2007-2014, using the SweMR linked to national population-based registries with high coverage. We identified 23'507 patients with primary invasive CM and included 1'162 patients with a diagnosis of type 2 diabetes mellitus in the study. The use of metformin pre-, peri-, and postdiagnostically was assessed among the patients. We showed that metformin use was consistently associated with significantly improved overall survival (OS) regardless of timing of treatment, although with no significant association on the melanoma-specific survival (MSS).In study III a cohort of 24'562 patients with a primary invasive CM diagnosed between 2006-2014 was identified, of whom 1'253 patients were using at least one of the six most common groups of H1-antihistamines. Drug use was assessed peri- and postdiagnostically. Desloratadine and loratadine use were associated with significantly improved MSS, in the analysis of peri- and postdiagnostic use, respectively, but no association on the MSS was seen for the other groups of antihistamines.In study IV, 252 patients with a diagnosis of metastatic (stage IV) CM and first-line treatment with immune checkpoint inhibitors (ICIs) or targeted therapy (TT) were identified from the records at the Department of Oncology/Skin Cancer Center, the Karolinska University Hospital, Sweden, between 1 January 2010 and 31 December 2017, and were followed until 31 March 2019. Patients receiving ICIs experienced significantly longer survival in terms of both overall- and progression-free survival (PFS) compared to patients receiving TTs, although not reaching statistical significance in the multivariable analysis for the latter. Several clinical markers were associated with therapy response and survival. Specifically, male sex and more advanced M-stage were associated with significantly lower response to both therapies and ICI, respectively. Furthermore, increasing lactase dehydrogenase levels were associated with shorter PFS and OS among both patient groups, and more advanced M-stage with lower OS and PFS among ICI-treated patients. Interestingly, decreasing albumin levels were associated with shorter PFS in patients treated with both therapies, lower OS for ICI-treated patients, and with a lower response rate to TT.In conclusion, we report that AI may be a useful tool for skin cancer diagnosis including CM, though further evaluation in prospective clinical trials is needed. Metformin, desloratadine, and loratadine are commonly used, cheap, and well- tolerated drugs that may have secondary antineoplastic effects, potentially contributing to improved survival among CM patients. Moreover, ICI and TT in first- line treatment of metastatic disease showed a survival benefit in a real-world setting. Finally, albumin may be a prognostic marker that needs further validation.List of scientific papersI. Isabelle Krakowski*, Jiyeong Kim*, Zhuo Ran Cai, Roxana Daneshjou, Jan Lapins, Hanna Eriksson, Anastasia Lykou, Eleni Linos. Human-AI interaction in skin cancer diagnosis: a systematic review and meta-analysis. NPJ Digit Med 2024; 7(1): 78. https://doi.org/10.1038/s41746-024-01031-wII. Isabelle Krakowski, Henrike Habel, Kari Nielsen, Christian Ingvar, Therese M L Andersson, Ada Girnita, Karin E Smedby, Hanna Eriksson. Association of metformin use and survival in patients with cutaneous melanoma and diabetes, Br J Dermatol 2023; 188(1): 32-40 https://doi.org/10.1093/bjd/ljac003III. Ildikó Fritz, Philippe Wagner, Matteo Bottai, Hanna Eriksson, Christian Ingvar, Isabelle Krakowski, Kari Nielsen, Håkan Olsson. Desloratadine and loratadine use associated with improved melanoma survival, Allergy 2020; 75(8): 2096- 9. https://doi.org/10.1111/all.14273IV. Isabelle Krakowski, Matteo Bottai, Henrike Häbel, Giuseppe Masucci, Ada Girnita, Karin E Smedby, Hanna Eriksson. Impact of modern systemic therapies and clinical markers on treatment outcome for metastatic melanoma in a real-world setting, J Eur Acad Dermatol Venereol 2021; 35(1): 105-15. https://doi.org/10.1111/jdv.16678</p
Exploring immunity, biodistribution, and toxicity of novel vaccines
No full textThis PhD thesis aimed to enhance the understanding of innate immunity, biodistribution, and safety through the study of modern immunostimulatory formulations, including an mRNA vaccine and an anti-CD40 monoclonal antibody (mAb), in non-human primate (NHP) models. The research was divided into three key studies:Paper I focused on the unmodified mRNA vaccine CVnCoV, which encodes the SARS-CoV-2 spike protein. The study examined early innate immune responses, biodistribution, and adaptive immune responses in NHPs following a low-dose regimen. This study highlighted the importance of optimizing vaccine doses to balance safety and efficacy, as lower doses resulted in limited antigen dissemination to lymph nodes, affecting the priming of adaptive immunity. However, additional doses were able to partly rescue suboptimal responses.Paper Il explored the development and evaluation of MAB273, a novel Fcy- receptor-independent agonistic anti-CD40 mAb, as an immunostimulatory adjuvant. The study assessed its binding and activation properties in vitro, as well as its safety, immune activation, pharmacokinetics, and tissue targeting in vivo. MAB273 effectively activated immune cells without relying on FcyR crosslinking. It also showed promise as an adjuvant, enhancing CD4 and CD8 T cell responses in both therapeutic and prophylactic vaccination models. MAB273 could thus represent a potential immunostimulatory adjuvant, deserving additional investigation in both therapeutic and preventive vaccination strategies.Paper III established age- and species-specific reference intervals for biochemical and hematological parameters in rhesus and cynomolgus macaques, which are critical for evaluating vaccine and drug safety. The study revealed that vaccination triggered transient inflammatory responses, with most parameters returning to baseline within 1-2 weeks. Key biomarkers, such as ALT and BUN, were important for assessing vaccination-induced effects on the liver and kidneys. This study also suggested a potential link between innate immune activation and adverse vaccine responses, warranting further investigation.Collectively, these studies contribute valuable insights into immunological mechanisms and safety, with implications for future vaccine development.List of scientific papersI. Lenart K, Hellgren F, Ols S, Yan X, Cagigi A, Cerveira RA, Winge I, Hanczak J, Mueller SO, Jasny E, Schwendt K, Rauch S, Petsch B, Loré K. A Third Dose of the Unmodified COVID-19 mRNA Vaccine CVnCoV Enhances Quality and Quantity of Immune Responses. Molecular Therapy - Methods & Clinical Development. 2022 Dec 8;27:309-323. https://doi.org/10.1016/j.omtm.2022.10.001II. Yan X, Ols S, Arcoverde Cerveira R, Lenart K, Hellgren F, Ye K, Cagigi A, Buggert M, Nimmerjahn F, Falkesgaard Højen J, Parera D, Pessara U, Fischer S, Loré K. Cell Targeting and Immunostimulatory Properties of a Novel Fcy- Receptor-Independent Agonistic Anti-CD40 Antibody in Rhesus Macaques. Cellular and Molecular Life Sciences. 2023 Jun 23;80(7):189. https://doi.org/10.1007/s00018-023-04828-2III. Yan X, Arcoverde Cerveira R, Ols S, Lenart K, Hellgren F, Engstrand O, Reinhardt A, Eriksson B, Loré K. Biochemical and Hematological Reference Intervals in Macaca mulatta and Macaca fascicularis: Implications for Vaccine and Drug Development. [Manuscript]</p
Soluble biomarkers to inform on pathophysiological mechanisms and for tailoring therapy in multiple sclerosis
No full textThis doctoral thesis explores the use of soluble biomarkers to elucidate pathophysiological mechanisms and inform tailored therapeutic strategies in multiple sclerosis (MS). The work is based on four studies that collectively address key aspects of MS diagnosis and treatment, focusing on B cell depleting therapies and their immunological effects.Firstly, in Paper I, we investigated the diagnostic accuracy of kappa free light chains (KFLC) compared to oligoclonal bands (OCB) in MS. The findings suggest that KFLC metrics, especially the KFLC intrathecal fraction and CSF KFLC, perform comparably to OCB, offering a potential alternative or complementary biomarker for MS diagnosis.In Paper II, we compared the effects of ocrelizumab (OCR) and rituximab (RTX) on immunoglobulin levels and safety in MS patients. Results indicate that OCR leads to a more pronounced reduction in immunoglobulin G (IgG) levels and CD19 numbers compared to low-dose RTX, with slightly higher rates of adverse events associated with OCR. Continuing with Paper III, we extended the analysis of RTX's impact on immunoglobulin levels over a longer follow-up period. A modest but significant decline in IgG and immunoglobulin M (IgM) levels was observed, with the decline influenced by factors such as previous treatments, age, and duration of RTX therapy.Lastly, in Paper IV, we focused on the immunological effects of RTX on T cell subsets. We revealed that RTX selectively reduces specific memory CD4 T cell populations, including Th17 and Th1.17 effector cells, which are implicated in MS inflammatory activity. The study also highlights the role of genetic factors, such as the DRB1*15:01 allele, in modulating T cell responses.Overall, this thesis underscores the utility of soluble biomarkers, particularly KFLC, in enhancing MS diagnostics. At the same time, IgG and IgM are highlighted to monitor the immunological impact of B cell depleting therapies. The findings contribute to a better understanding of MS pathophysiology and suggest potential avenues for optimising treatment strategies.List of scientific papersShared authorship #I. Duell F, Evertsson B, Al Nimer F, Sandin Å, Olsson D, Olsson T, Khademi M, Hietala MA, Piehl F, Hansson M. Diagnostic accuracy of intrathecal kappa free light chains compared with OCBs in MS. Neurol Neuroimmunol Neuroinflamm. 2020 Jun 11;7(4):e775. https://doi.org/10.1212/NXI.0000000000000775II. Evertsson B#, Hoyt T#, Christensen A, Nimer FA, Foley J#, Piehl F#. A comparative study of tolerability and effects on immunoglobulin levels and CD19 cell counts with ocrelizumab vs low dose of rituximab in multiple sclerosis. Mult Scler J Exp Transl Clin. 2020 Oct 12;6(4):2055217320964505. https://doi.org/10.1177/2055217320964505III. Hallberg S#, Evertsson B#, Lillvall E, Boremalm M, de Flon P, Wang Y, Salzer J, Lycke J, Fink K, Frisell T, Al Nimer F, Svenningsson A. Hypogammaglobulinaemia during rituximab treatment in multiple sclerosis: A Swedish cohort study. Eur J Neurol. 2024 Aug;31(8):e16331. Epub 2024 May 25. https://doi.org/10.1111/ene.16331IV. Evertsson B, Ruffin N, Theorell J, Huang J, Asplund Högelin K, Khademi K, Piehl F, Nimer FA. B cell depletion has a distinct influence on T cell immunity by decreasing effector memory CD4 Th17 and Th1.17 subsets in multiple sclerosis. [Manuscript]</p
The role of atypical ubiquitin chains in intestinal homeostasis and colorectal cancer
No full textAbstractUbiquitination is a post-translational modification that is essential for protein regulation in the cell and is involved in virtually all cellular processes. Although ubiquitination has been studied for more than 40 years, there are still many unknowns, including the role of atypical ubiquitin chains, a type of ubiquitin modification that is underrepresented in cells. The functions of these atypical chains, the physiological and pathological processes involved, and the underlying molecular mechanisms are still poorly characterized. In this Ph.D. project, I aim to narrow this knowledge gap by studying Trabid, a deubiquitinating enzyme specific for atypical ubiquitin chains, and whose own functions remain enigmatic. Our first studies characterizing Trabid-deficient mice suggested that Trabid, and in turn, most likely, atypical ubiquitin chains, could be involved in the regulation of the intestinal Stem cell niche. Intestinal Stem cells are important for intestinal homeostasis, being involved not only in the renewal of intestinal epithelial cells under physiological conditions but also in the repair of intestinal damage. In addition, pathological hyperplasia of intestinal Stem cells can lead to the development of colorectal cancer. Thus, this Ph.D. project is of relevance not only for the ubiquitin field but also for regenerative and cancer therapies.</p