Patients suffering from dystrophic epidermolysis bullosa are prone to developing autoantibodies against skin proteins: A longitudinal confirmational study.

Epidermolysis bullosa (EB) is a heritable skin blistering disease caused by variants in genes coding for proteins that secure cell-cell adhesion and attachment of the epidermis to the dermis. Interestingly, several proteins involved in inherited EB are also associated with autoimmune blistering diseases (AIBD). In this study, we present a long-term follow-up of 15 patients suffering from recessive dystrophic or junctional EB. From these patients, 62 sera were analysed for the presence of autoantibodies associated with AIBD. We show that patients suffering from recessive dystrophic EB (RDEB) are more susceptible to developing autoantibodies against skin proteins than patients suffering from junctional EB (70% vs. 20%, respectively). Interestingly, no correlation with age was observed. Most patients showed reactivity to Type XVII collagen/linear IgA bullous dermatosis autoantigen (n = 5; 33%), followed by BP230 (n = 4; 27%), Type VII collagen (n = 4; 27%) and laminin-332 (n = 1; 7%). The pathogenicity of these autoantibodies remains a subject for future experiments.

Single-arm trials supporting the approval of anticancer medicinal products in the European Union: contextualization of trial results and observed clinical benefit.

BACKGROUND: Single-arm trials (SATs) can sometimes be used to support marketing authorization of anticancer medicinal products in the European Union. The level and durability of antitumor activity of the product as well as context are important aspects to determine the relevance of trial results. The aim of this study is to provide details on the contextualization of trial results and to evaluate the magnitude of benefit of medicinal products approved based on SATs. MATERIALS AND METHODS: We focused on anticancer medicinal products for solid tumors approved on the basis of SAT results (2012-2021). Data were retrieved from European public assessment reports and/or published literature. The benefit of these medicinal products was evaluated via the European Society for Medical Oncology (ESMO)-Magnitude of Clinical Benefit Scale (MCBS). RESULTS: Eighteen medicinal products were approved based on 21 SATs-few medicinal products were supported by >1 SAT. For the majority of clinical trials, a clinically relevant treatment effect was (pre)specified (71.4%) and most often an accompanying sample size calculation was provided. For 10 studies, each testing a different medicinal product, a justification for the threshold for a clinically relevant treatment effect could be identified. At least 12 out of 18 applications included information to facilitate the contextualization of trial results, including six supportive studies. Of the pivotal SATs analyzed (n = 21), three were assigned an ESMO-MCBS score of 4, which corresponds to 'substantial' benefit. CONCLUSIONS: The clinical relevance of the treatment effects shown by medicinal products for solid tumors tested in SATs is dependent on the effect size and context. To better facilitate regulatory decision making, prespecifying and motivating a clinically relevant effect and aligning the sample size to that effect is important. External controls may facilitate in the contextualization process, but the associated limitations must be addressed.

Individual patient data to allow a more elaborated comparison of trial results with real-world outcomes from first-line immunotherapy in NSCLC.

BACKGROUND: Many studies have compared real-world clinical outcomes of immunotherapy in patients with metastatic non-small cell lung cancer (NSCLC) with reported outcomes data from pivotal trials. However, any differences observed could be only limitedly explored further for causation because of the unavailability of individual patient data (IPD) from trial participants. The present study aims to explore the additional benefit of comparison with IPD. METHODS: This study compares progression free survival (PFS) and overall survival (OS) of metastatic NSCLC patients treated with second line nivolumab in real-world clinical practice (n = 141) with IPD from participants in the Checkmate-057 clinical trial (n = 292). Univariate and multivariate Cox proportional hazards models were used to construct HRs for real-world practice versus clinical trial. RESULTS: Real-world patients were older (64 vs. 61 years), had more often ECOG PS ≥ 2 (5 vs. 0%) and were less often treated with subsequent anti-cancer treatment (28.4 vs. 42.5%) compared to trial patients. The median PFS in real-world patients was longer (3.84 (95%CI: 3.19-5.49) vs 2.30 (2.20-3.50) months) and the OS shorter than in trial participants (8.25 (6.93-13.2) vs. 12.2 (9.90-15.1) months). Adjustment with available patient characteristics, led to a shift in the hazard ratio (HR) for OS, but not for PFS (HRs from 1.13 (0.88-1.44) to 1.07 (0.83-1.38), and from 0.82 (0.66-1.03) to 0.79 (0.63-1.00), respectively). CONCLUSIONS: This study is an example how IPD from both real-world and trial patients can be applied to search for factors that could explain an efficacy-effectiveness gap. Making IPD from clinical trials available to the international research community allows this.

Long-term survival of patients with advanced melanoma treated with BRAF-MEK inhibitors.

Recent results of patients with advanced melanoma treated with first-line BRAF-MEK inhibitors in clinical trials showed 5-year survival in one-third of patients with a median overall survival (OS) of more than 2 years. This study aimed to investigate these patients' real-world survival and identify the characteristics of long-term survivors. The study population consisted of patients with advanced cutaneous melanoma with a BRAF-V600 mutated tumor who were treated with first-line BRAF-MEK inhibitors between 2013 and 2017. Long-term survival was defined as a minimum OS of 2 years from start therapy. The median progression-free survival (mPFS) and median OS (mOS) of real-world patients ( n = 435) were respectively 8.0 (95% CI, 6.8-9.4) and 11.7 (95% CI, 10.3-13.5) months. Two-year survival was reached by 28% of the patients, 22% reached 3-year survival and 19% reached 4-year survival. Real-world patients often had brain metastases (41%), stage IV M1c disease (87%), ECOG PS ≥2 (21%), ≥3 organ sites (62%) and elevated LDH of ≥250 U/I (49%). Trial-eligible real-world patients had an mOS of 17.9 months. Patients surviving more than 2 years ( n = 116) more often had an ECOG PS ≤1 (83%), normal LDH (60%), no brain metastases (60%), no liver metastases (63%) and <3 organ sites (60%). Long-term survival of real-world patients treated with first-line BRAF-MEK inhibitors is significantly lower than that of trial patients, which is probably explained by poorer baseline characteristics of patients treated in daily practice. Long-term survivors generally had more favorable characteristics with regard to age, LDH level and metastatic sites, compared to patients not reaching long-term survival.

Palbociclib dose reductions and the effect on clinical outcomes in patients with advanced breast cancer.

BACKGROUND: This study aimed to provide insights into the real-world use of palbociclib, dose reductions, and drug effectiveness in (older) patients with advanced breast cancer (BC). PATIENTS AND METHODS: Patients with advanced BC treated with palbociclib from 2017 to 2020 were included. The Kaplan-Meier method was used to calculate time to next treatment (TTNT) and overall survival (OS) for patients with or without dose reductions. These clinical outcomes were also compared in subgroup analyses for older patients (≥70 years) and younger patients (<70 years) and for patients discontinuing palbociclib early (<4 administrations). RESULTS: A total of 598 patients with advanced BC were included, with a median age of 64 years. Palbociclib dose reductions occurred in 33% of all patients. Early discontinuation of palbociclib without dose reductions occurred in 23% of the patients. Patients who required a palbociclib dose reduction were older (median age 67 years vs. 63 years). Patients with dose reductions had a significantly higher TTNT of 16.9 vs. 11.4 months (p < 0.001) and median OS of 29.7 vs. 21.9 months (p = 0.003) compared to patients without dose reductions. The TTNT in older patients was significantly longer (16.9 vs. 11.6 months, p = 0.013) than younger patients, but OS was similar (20.7 vs. 26.7 months, p = 0.051). CONCLUSION: Palbociclib dose reductions occurred in real-world practice similarly to the PALOMA-3 trial. Patients with dose reductions had no poorer outcomes compared to patients not requiring a dose reduction. Older patients treated with palbociclib had more frequent dose reductions, but this did not appear to affect OS.

Novel insights into the epidemiology of epidermolysis bullosa (EB) from the Dutch EB Registry: EB more common than previously assumed?

BACKGROUND: Epidermolysis bullosa (EB) is a heterogeneous group of rare and incurable genetic disorders characterized by fragility of the skin and mucosae, resulting in blisters and erosions. Several epidemiological studies in other populations have been carried out, reporting varying and sometimes inconclusive figures, highlighting the need for standardized epidemiological analyses in well-characterized cohorts. OBJECTIVES: To evaluate the epidemiological data on EB in the Netherlands, extracted from the molecularly well-characterized cohort in the Dutch EB Registry. METHODS: In this observational study all EB-patients that were based in the Netherlands and captured in the Dutch EB Registry between 1988 and 2018 were included. The epidemiological outcomes were based on complete diagnostic data (clinical features, immunofluorescence, electron microscopy and mutation analysis), with longitudinal follow-up. RESULTS: A total of 464 EB-patients (287 families) were included. The incidence and point-prevalence of EB in the Netherlands were 41.3 per million live births and 22.4 per million population, respectively. EB Simplex (EBS), Junctional EB (JEB), Dystrophic EB (DEB) and Kindler EB were diagnosed in 45.7%, 18.8%, 34.7% and 0.9% of the EB-patients, respectively, with an incidence and point-prevalence of 17.5 and 11.9 (EBS), 9.3 and 2.1 (JEB), 14.1 and 8.3 (DEB), 0.5 and 0.2 (Kindler EB). In 90.5% of the EB-patients the diagnosis was genetically confirmed. During the investigated time period 73 EB-patients died, 72.6% of whom as a direct consequence of their EB. CONCLUSION: The epidemiological outcomes of EB in the Netherlands are high, attributed to a high detection rate in a well-organized set-up, indicating that EB might be more common than previously assumed. These epidemiological data help to understand the extensive need for (specialized) medical care of EB-patients and is invaluable for the design and execution of therapeutic trials. This study emphasizes the importance of thorough reporting systems and registries worldwide.

Amelioration of junctional epidermolysis bullosa due to exon skipping.

Mutations in the COL17A1 gene lead to the genetic blistering disorder junctional epidermolysis bullosa generalized intermediate type (JEB-gen-intermed). Antisense oligonucleotide-mediated exon skipping is a strategy that aims to skip the mutation-containing exon and thereby produce a smaller but functional protein. COL17A1 is an interesting candidate, as 53 of the 55 exons (96%) can be skipped without disturbing the reading frame. Information on the functionality of the shortened protein product is important in order to obtain support for this therapeutic strategy. Here we report a patient with JEB-gen-intermed with amelioration of the phenotype due to exon 49 skipping by two distinct mechanisms - premature termination codon-induced exon skipping and revertant mosaicism - both of which induced skipping of the same exon. The patient was compound heterozygous for two inherited COL17A1 mutations, a frameshift mutation in exon 18 (c.1490_1491delinsT, p.Ala497Valfs*23) and a nonsense mutation in exon 49 (c.3487G>T, p.Glu1163Ter). Upon clinical examination, skin patches were found that were resistant to blister formation. In these patches, naturally corrected cells were present that harboured an additional splice-site mutation, c.3419-1G>T, resulting in skipping of the mutation-containing exon 49. This natural gene therapy phenomenon shows that type XVII collagen with residues 1140-1169 deleted is largely functional. In addition, in affected skin cells a low level of exon 49 skipping was observed. Our results support the notion that skipping of a mutated in-frame exon in COL17A1 ameliorates the phenotype.

Somatic mosaicism for the COL7A1 mutation p.Gly2034Arg in the unaffected mother of a patient with dystrophic epidermolysis bullosa pruriginosa.

Dystrophic epidermolysis bullosa (DEB) is a heritable blistering disorder caused by mutations in the type VII collagen gene, COL7A1. Although revertant mosaicism is well known in DEB, 'forward' somatic mosaicism, in which a pathogenic mutation arises on a wild-type (WT) background, extending beyond the germ cells, has not been reported. It is therefore unknown what proportion of sporadic dominant DEB (DDEB) cases result from de novo mutations or somatic mosaic parents. In the clinically unaffected mother of a patient with DDEB pruriginosa due to the p.Gly2034Arg mutation, we identified the p.Gly2034Arg mutation in a proportion of lymphocytes and skin cells (mutational load 10-25%). Our data emphasize that forward mosaicism occurs in DDEB and highlight that mutation analysis should always be performed in the parents of sporadic DDEB patients to confirm the de novo status of the mutation. Ultimately, this will reveal the frequency of true de novo mutations and somatic mosaicism in parents, which has important implications for genetic counselling. Our data indicate that the threshold of mutant type VII procollagen to develop DDEB must be higher than 10-25%, which provides a rationale for therapeutic approaches aimed at increasing the WT : mutant type VII collagen ratio.

Adhesive stripping to remove epidermis in junctional epidermolysis bullosa for revertant cell therapy.

BACKGROUND: Replacing mutant skin in epidermolysis bullosa (EB) by epithelial sheets of transduced autologous keratinocytes is the essential surgical step of ex vivo gene therapy. The same applies for revertant cell therapy in which epithelial sheets of revertant autologous keratinocytes are used. Revertant cells can be found in patches of normal skin in patients with junctional EB (JEB) due to revertant mosaicism caused by in vivo reversions. OBJECTIVES: To develop a technique of adhesive tape stripping as a method for epidermis removal to prepare the acceptor site for revertant cell therapy in a patient with revertant mosaic JEB. METHODS: We performed revertant cell therapy on a patient with mosaic type XVII collagen-deficient non-Herlitz JEB. Skin biopsies were taken from revertant skin on the wrist. Graft production took place on a 3T3-J2 feeder layer resulting in two 6 x 7 cm grafts. An innovative method that uses the pathological plane of least resistance of JEB skin was developed to prepare the acceptor site. A polyacrylate adhesive plaster was placed on the skin and then pulled off with the epidermis. RESULTS: The epidermis was easily removed with the plaster. The skin separated at the level of the lamina lucida, leaving a bloodless wound bed of naked lamina densa. Transplantation was successful; the acceptor site healed without scarring. However, blistering could be provoked. The functional repair was not achieved due to the low percentage of revertant cells in the graft. CONCLUSIONS: We conclude that adhesive stripping is a simple, effective and almost painless procedure for removing epidermis for ex vivo cell therapy in EB.

Localized and generalized forms of blistering in junctional epidermolysis bullosa due to COL17A1 mutations in the Netherlands.

BACKGROUND: Mutations in the gene COL17A1 coding for type XVII collagen cause non-Herlitz junctional epidermolysis bullosa (nH-JEB). OBJECTIVES: Here we give an overview of the genotype-phenotype correlation in 12 patients from the Netherlands with type XVII collagen-deficient nH-JEB. PATIENT AND METHODS: Family and personal history and clinical presentation were recorded from each patient, and skin biopsies of intact and bullous skin were taken for immunofluorescence and electron microscopy. The mutations were identified by analysing the patient's DNA isolated from peripheral blood cells. RESULTS: DNA analysis identified five novel deletions: 1284delA, 1365delC, 3236delT, 3600-3601delCT and 4425delT. Interestingly, we identified a new patient, homozygous for 4425delT, with an exceptionally mild blistering phenotype. All together, three patients had more localized blistering confined to hands, lower legs and face, absent or very mild nail dystrophy, normal primary hair and sparse secondary hair. Nine patients had generalized blistering, nail dystrophy, sparse primary and absent secondary hair. All 12 patients had amelogenesis imperfecta (enamel pitting). Immunofluorescence (IF) antigen mapping with monoclonal antibodies 1A8C and 1D1 that bind to type XVII collagen, but not to its 97-kDa fragment was completely negative in patients with generalized blistering, whereas reduced in patients with localized blistering. CONCLUSIONS: Our data reveal that in patients with COL17A1 mutations a localized nH-JEB phenotype can be differentiated from a generalized nH-JEB phenotype by IF antigen mapping. The data are important for genetic counselling at early age when the clinical phenotype is not yet clear.

Features of epidermolysis bullosa simplex due to mutations in the ectodomain of type XVII collagen.

BACKGROUND: Mutations in COL17A1, coding for type XVII collagen, cause junctional epidermolysis bullosa with an ultrastructural plane of cleavage through the lamina lucida of the epidermal basement membrane. OBJECTIVES: To identify the COL17A1 mutations in a child with reduced type XVII collagen expression and intraepidermal blister formation. PATIENT AND METHODS: Protein expression and level of tissue separation were studied by immunofluorescence and electron microscopy. The mutations were identified by analysing the patient's DNA and mRNA. RESULTS: Immunofluorescence microscopy performed on nonlesional skin demonstrated absence of the type XVII collagen endodomain and presence, although reduced, of the shed ectodomain. Electron microscopy showed that the plane of cleavage was through the basal cells, not through the lamina lucida. Two heterozygous mutations were identified in COL17A1: a new 3'-acceptor splice-site mutation in intron 21 (1877-2A-->C), and a deletion in exon 48 (3432delT). The splice-site mutation in intron 21 results in alternative transcripts of which two are in-frame, with deletions of the first nine codons of exon 22 and the entire exon 22, respectively. By Western blot analysis, a type XVII collagen molecule was detected that was slightly smaller than normal. CONCLUSIONS: Occasionally mutations in the COL17A1 gene may result in split levels suggesting epidermolysis bullosa simplex rather than junctional epidermolysis bullosa.

A very mild form of non-Herlitz junctional epidermolysis bullosa: BP180 rescue by outsplicing of mutated exon 30 coding for the COL15 domain.

Mutations in the gene COL17A1 cause non-Herlitz junctional epidermolysis bullosa. Here, we describe a patient who, despite two heterozygous mutations in COL17A1, has an extremely mild form of the disease missing most of the characteristic clinical features. DNA analysis revealed a frame-shift mutation 3432delT and a nonsense mutation 2356C-->T (Q751X). cDNA analysis showed that the deleterious effect of the latter mutation was skirted by deleting the premature termination codon containing exon 30. In this way, the reading frame was restored, resulting in a 36 nucleotides shorter mRNA transcript. Immunoblot analysis showed expression of the 180-kDa bullous pemphigoid antigen (BP180) with a slightly higher SDS-PAGE mobility, in line with the deletion of 12 amino acids from the COL15 domain. Immunofluorescence of skin sections showed diminished, but correctly localised expression of BP180, and this, in concert with the mild clinical phenotype, suggests that this COL15 mutated BP180 is still partly functional.