A neutral comparison of statistical methods for analyzing longitudinally measured ordinal outcomes in rare diseases.

Ordinal data in a repeated measures design of a crossover study for rare diseases usually do not allow for the use of standard parametric methods, and hence, nonparametric methods should be considered instead. However, only limited simulation studies in settings with small sample sizes exist. Therefore, starting from an Epidermolysis Bullosa simplex trial with the above-mentioned design, a rank-based approach using the R package nparLD and different generalized pairwise comparisons (GPC) methods were compared impartially in a simulation study. The results revealed that there was not one single best method for this particular design, because a trade-off exists between achieving high power, accounting for period effects, and for missing data. Specifically, nparLD as well as the unmatched GPC approaches do not address crossover aspects, and the univariate GPC variants partly ignore the longitudinal information. The matched GPC approaches, on the other hand, take the crossover effect into account in the sense of incorporating the within-subject association. Overall, the prioritized unmatched GPC method achieved the highest power in the simulation scenarios, although this may be due to the specified prioritization. The rank-based approach yielded good power even at a sample size of N = 6 $N=6$ , whereas the matched GPC method could not control the type I error.

Statistical recommendations for count, binary, and ordinal data in rare disease cross-over trials.

BACKGROUND: Recommendations for statistical methods in rare disease trials are scarce, especially for cross-over designs. As a result various state-of-the-art methodologies were compared as neutrally as possible using an illustrative data set from epidermolysis bullosa research to build recommendations for count, binary, and ordinal outcome variables. For this purpose, parametric (model averaging), semiparametric (generalized estimating equations type [GEE-like]) and nonparametric (generalized pairwise comparisons [GPC] and a marginal model implemented in the R package nparLD) methods were chosen by an international consortium of statisticians. RESULTS: It was found that there is no uniformly best method for the aforementioned types of outcome variables, but in particular situations, there are methods that perform better than others. Especially if maximizing power is the primary goal, the prioritized unmatched GPC method was able to achieve particularly good results, besides being appropriate for prioritizing clinically relevant time points. Model averaging led to favorable results in some scenarios especially within the binary outcome setting and, like the GEE-like semiparametric method, also allows for considering period and carry-over effects properly. Inference based on the nonparametric marginal model was able to achieve high power, especially in the ordinal outcome scenario, despite small sample sizes due to separate testing of treatment periods, and is suitable when longitudinal and interaction effects have to be considered. CONCLUSION: Overall, a balance has to be found between achieving high power, accounting for cross-over, period, or carry-over effects, and prioritizing clinically relevant time points.

MicroRNA-200b-mediated reversion of a spectrum of epithelial-to-mesenchymal transition states in recessive dystrophic epidermolysis bullosa squamous cell carcinomas.

BACKGROUND: Cutaneous squamous cell carcinoma (SCC) is the leading cause of death in patients with recessive dystrophic epidermolysis bullosa (RDEB). However, the survival time from first diagnosis differs between patients; some tumours spread particularly fast, while others may remain localized for years. As treatment options are limited, there is an urgent need for further insights into the pathomechanisms of RDEB tumours, to foster therapy development and support clinical decision-making. OBJECTIVES: To investigate differences in RDEB tumours of diverging aggressiveness at the molecular and phenotypic level, with a particular focus on epithelial-to-mesenchymal (EMT) transition states and thus microRNA-200b (miR-200b) as a regulator. METHODS: Primary RDEB-SCC keratinocyte lines were characterized with respect to their EMT state. For this purpose, cell morphology was classified and the expression of EMT markers analysed using immunofluorescence, flow cytometry, semi-quantitative reverse transcriptase polymerase chain reaction and Western blotting. The motility of RDEB-SCC cells was determined and conditioned medium of RDEB-SCC cells was used to treat endothelial cells in an angiogenesis assay. In addition, we mined previously generated microRNA (miRNA) profiling data to identify a candidate with potential therapeutic relevance and performed transient miRNA transfection studies to investigate the candidate's ability to reverse EMT characteristics. RESULTS: We observed high variability in EMT state in the RDEB-SCC cell lines, which correlated with in situ analysis of two available patient biopsies and respective clinical disease course. Furthermore, we identified miR-200b-3p to be downregulated in RDEB-SCCs, and the extent of deregulation significantly correlated with the EMT features of the various tumour lines. miR-200b-3p was reintroduced into RDEB-SCC cell lines with pronounced EMT features, which resulted in a significant increase in epithelial characteristics, including cell morphology, EMT marker expression, migration and angiogenic potential. CONCLUSIONS: RDEB-SCCs exist in different EMT states and the level of miR-200b is indicative of how far an RDEB-SCC has gone down the EMT path. Moreover, the reintroduction of miR-200b significantly reduced mesenchymal features.

Type VII Collagen Deficiency in the Oncogenesis of Cutaneous Squamous Cell Carcinoma in Dystrophic Epidermolysis Bullosa.

Dystrophic epidermolysis bullosa is a rare genetic skin disorder caused by COL7A1 sequence variations that result in type VII collagen deficits and cutaneous and extracutaneous manifestations. One serious complication of dystrophic epidermolysis bullosa is cutaneous squamous cell carcinoma, a leading driver of morbidity and mortality, especially among patients with recessive dystrophic epidermolysis bullosa. Type VII collagen deficits alter TGFß signaling and evoke multiple other cutaneous squamous cell carcinoma progression-promoting activities within epidermal microenvironments. This review examines cutaneous squamous cell carcinoma pathophysiology in dystrophic epidermolysis bullosa with a focus on known oncogenesis pathways at play and explores the idea that therapeutic type VII collagen replacement may reduce cutaneous squamous cell carcinoma risk.

Heterogeneity of reported outcomes in epidermolysis bullosa clinical research: a scoping review as a first step towards outcome harmonization.

BACKGROUND: Epidermolysis bullosa (EB) is a rare, genetically and clinically heterogeneous group of skin fragility disorders. No cure is currently available, but many novel and repurposed treatments are upcoming. For adequate evaluation and comparison of clinical studies in EB, well-defined and consistent consensus-endorsed outcomes and outcome measurement instruments are necessary. OBJECTIVES: To identify previously reported outcomes in EB clinical research, group these outcomes by outcome domains and areas and summarize respective outcome measurement instruments. METHODS: A systematic literature search was performed in the databases MEDLINE, Embase, Scopus, Cochrane CENTRAL, CINAHL, PsycINFO and trial registries covering the period between January 1991 and September 2021. Studies were included if they evaluated a treatment in a minimum of three patients with EB. Two reviewers independently performed the study selection and data extraction. All identified outcomes and their respective instruments were mapped onto overarching outcome domains. The outcome domains were stratified according to subgroups of EB type, age group, intervention, decade and phase of clinical trial. RESULTS: The included studies (n = 207) covered a range of study designs and geographical settings. A total of 1280 outcomes were extracted verbatim and inductively mapped onto 80 outcome domains and 14 outcome areas. We found a steady increase in the number of published clinical trials and outcomes reported over the past 30 years. The included studies mainly focused on recessive dystrophic EB (43%). Wound healing was reported most frequently across all studies and referred to as a primary outcome in 31% of trials. Great heterogeneity of reported outcomes was observed within all stratified subgroups. Moreover, a diverse range of outcome measurement instruments (n = 200) was identified. CONCLUSIONS: We show substantial heterogeneity in reported outcomes and outcome measurement instruments in EB clinical research over the past 30 years. This review is the first step towards harmonization of outcomes in EB, which is necessary to expedite the clinical translation of novel treatments for patients with EB.

Recessive Dystrophic Epidermolysis bullosa due to Hemizygous 40 kb Deletion of COL7A1 and the Proximate PFKFB4 Gene Focusing on the Mutation c.425A>G Mimicking Homozygous Status

Background: Dystrophic Epidermolysis bullosa (DEB) is a rare inherited mechanobullous disease characterised by the hyperfragility of the skin and mucous membranes. It is (typically) caused by (loss-of-function) mutations in the COL7A1 gene that impair the formation of collagen type VII, which represents the major constituent of anchoring fibrils within the basement membrane zone of epithelialised tissues. In a 4-year-old patient diagnosed with the clinical features of recessive DEB, genotyping via Next-Generation EB Panel Sequencing initially revealed the homozygosity of the maternal c.425A>G mutation, while the paternal heterozygosity in exon 3 was lacking. This genetic profile suggested incongruent gene transmission due to uniparental isodisomy (UPD) or the occurrence of a hemizygous deletion of unknown size. Methods: Thus, the EB panel sequencing of genomic DNA, followed by a paternity test and analysis of microsatellite markers, as well as multiplex ligation-dependent probe amplification (MLPA) copy number analysis using patient and parental DNA, were performed. Results: This approach revealed a paternally derived hemizygous deletion spanning from exon 3 to exon 118. Linear amplification-mediated PCR (LAM-PCR) determined the breaking points within intron 2 of the COL7A1 gene, comprising a 40kb segment within intron 1 of the adjacent PFKFB4 gene. Conclusion: This report highlights the relevance of advanced molecular profiling to determine new/exceptional/unusual genotypes and the accurate mode of genetic transmission in DEB.

Equivocal Longitudinal Melanonychia in a 3-Year-Old Caucasian Girl: Rapid Evolution and Fading during Digital Dermoscopy Follow-Up.

Introduction: Nail matrix nevi (NMN) in pediatric patients manifest as longitudinal melanonychia (LM) and can share clinical, dermoscopic, and histopathological characteristics with subungual melanoma. Equivocal findings in childhood LM may reflect dynamic processes during the natural life cycle of NMN in children. Case Presentation: We present a case of a heavily pigmented LM with equivocal clinical and dermoscopic findings in a 3-year-old Caucasian girl, which exhibited signs of evolution, maturation, and almost complete involution within a short time period during digital follow-up, attributed to the natural course of NMN. Discussion/Conclusion: Considering the rarity of subungual melanoma in childhood, our case underlines the significance of clinical and digital dermoscopy follow-up in the evaluation of childhood LM in order to avoid unnecessary biopsies and potential permanent nail dystrophy.

Impact of low-dose calcipotriol ointment on wound healing, pruritus and pain in patients with dystrophic epidermolysis bullosa: A randomized, double-blind, placebo-controlled trial.

BACKGROUND: Wound management is a critical factor when treating patients with the inherited skin fragility disease dystrophic epidermolysis bullosa (DEB). Due to genetic defects in structural proteins, skin and mucous epithelia are prone to blistering and chronic wounding upon minor trauma. Furthermore, these wounds are commonly associated with excessive pruritus and predispose to the development of life-threatening squamous cell carcinomas, underscoring the unmet need for new therapeutic options to improve wound healing in this patient cohort. Vitamin D3 is acknowledged to play an important role in wound healing by modulating different cellular processes that impact epidermal homeostasis and immune responses. In this study, we evaluate the safety and efficacy of low-dose calcipotriol, a vitamin D3 analogue, in promoting wound healing and reducing itch and pain in patients with DEB. METHODS: Eligible DEB patients, aged ≥ 6 years and with a known mutation in the COL7A1 gene, were recruited to a placebo-controlled, randomized, double blind, cross-over phase II monocentric clinical trial. Patients were required to have at least two wounds with a minimum size of 6 cm2 per wound. The primary objective was to evaluate efficacy of daily topical application of a 0.05 µg/g calcipotriol ointment in reducing wound size within a 4-week treatment regimen. Secondary objectives were to assess safety, as well as the impact of treatment on pruritus, pain, and bacterial wound colonization in these patients. RESULTS: Six patients completed the clinical trial and were included into the final analysis. Topical low-dose calcipotriol treatment led to a significant reduction in wound area at day 14 compared to placebo (88.4% vs. 65.5%, P < 0.05). Patients also reported a significant reduction of pruritus with calcipotriol ointment compared to placebo over the entire course of the treatment as shown by itch scores of 3.16 vs 4.83 (P < 0.05) and 1.83 vs 5.52 (P < 0.0001) at days 14 and 28, respectively. Treatment with low-dose calcipotriol did not affect serum calcium levels and improved the species richness of the wound microbiome, albeit with no statistical significance. CONCLUSIONS: Our results show that topical treatment with low-dose calcipotriol can accelerate wound closure and significantly reduces itch, and can be considered a safe and readily-available option to improve local wound care in DEB patients. Trial Registration EudraCT: 2016-001,967-35. Registered 28 June 2016, https://www.clinicaltrialsregister.eu/ctr-search/trial/2016-001967-35/AT.

Clinical trial of ABCB5+ mesenchymal stem cells for recessive dystrophic epidermolysis bullosa.

BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is a rare, devastating, and life-threatening inherited skin fragility disorder that comes about due to a lack of functional type VII collagen, for which no effective therapy exists. ABCB5+ dermal mesenchymal stem cells (ABCB5+ MSCs) possess immunomodulatory, inflammation-dampening, and tissue-healing capacities. In a Col7a1-/- mouse model of RDEB, treatment with ABCB5+ MSCs markedly extended the animals' lifespans.METHODSIn this international, multicentric, single-arm, phase I/IIa clinical trial, 16 patients (aged 4-36 years) enrolled into 4 age cohorts received 3 i.v. infusions of 2 × 106 ABCB5+ MSCs/kg on days 0, 17, and 35. Patients were followed up for 12 weeks regarding efficacy and 12 months regarding safety.RESULTSAt 12 weeks, statistically significant median (IQR) reductions in the Epidermolysis Bullosa Disease Activity and Scarring Index activity (EBDASI activity) score of 13.0% (2.9%-30%; P = 0.049) and the Instrument for Scoring Clinical Outcome of Research for Epidermolysis Bullosa clinician (iscorEB­c) score of 18.2% (1.9%-39.8%; P = 0.037) were observed. Reductions in itch and pain numerical rating scale scores were greatest on day 35, amounting to 37.5% (0.0%-42.9%; P = 0.033) and 25.0% (-8.4% to 46.4%; P = 0.168), respectively. Three adverse events were considered related to the cell product: 1 mild lymphadenopathy and 2 hypersensitivity reactions. The latter 2 were serious but resolved without sequelae shortly after withdrawal of treatment.CONCLUSIONThis trial demonstrates good tolerability, manageable safety, and potential efficacy of i.v. ABCB5+ MSCs as a readily available disease-modifying therapy for RDEB and provides a rationale for further clinical evaluation.TRIAL REGISTRATIONClinicaltrials.gov NCT03529877; EudraCT 2018-001009-98.FUNDINGThe trial was sponsored by RHEACELL GmbH & Co. KG. Contributions by NYF and MHF to this work were supported by the NIH/National Eye Institute (NEI) grants RO1EY025794 and R24EY028767.

COVID-19-Induced Reduction in Primary Melanoma Diagnoses: Experience from a Dermatopathology Referral Center.

The collateral damage caused by COVID-19 pandemic-associated public health and governmental measures on patient care has been increasingly assessed in various oncological and non-oncological clinical settings. In order to investigate potential adverse effects in the field of melanoma the present study analyzed the characteristics of primary melanoma diagnoses at an Austrian dermato-pathological referral center before, during, and after the first coronavirus-related lockdown in March 2020. As suspected, we found significant temporary reductions in the number of newly diagnosed melanomas in 2020 compared to previous years, in particular, during the first lockdown period.

The natural history of laryngo-onycho-cutaneous syndrome: A case series of six pediatric patients and literature review.

BACKGROUND/OBJECTIVES: Laryngo-onycho-cutaneous syndrome (LOC) is a rare subtype of junctional epidermolysis bullosa (JEB), featuring aberrant granulation tissue formation in the skin, larynx, and eyes. So far, three mutations including the specific (founder) mutation in exon 39 of LAMA3 (c.151dup) have been identified, but sparse data exists regarding the natural history, the genotype-phenotype correlation, and its differentiation from other JEB types. METHODS: We reviewed our pediatric EB database to identify English children with clinical and genetically diagnosed LOC within the last 15 years. Their demographic, clinical, and laboratory data were examined. We searched three databases for case reports of LOC between January 1986 and November 2020 and extracted clinical and molecular details. RESULTS: We identified 6 LOC patients, all female (mean age 5.4 years). Periungual hypergranulation and skin fragility were the earliest presenting signs (0-3 months), followed by laryngeal stenosis, symblepharon (mean onset 10.7 and 11.8 months, respectively), and dental abnormalities. Five children developed anemia at an average of 19.2 months. We identified 22 published studies in English with 31 cases. CONCLUSIONS: This study delineates the disease course of LOC and highlights the overlap with some forms of JEB. Classical signs/symptoms including anemia appear early in life. Genetic analysis revealed three new LOC-associated variants and underscores the finding that interpretation of skin immunolabeling and molecular diagnostics can be challenging. We provide recommendations on management of this complex syndrome.