We present two illustrative cases highlighting diagnostic, surveillance and management complexities of TP53 pathogenic variants (PVs). Case 1 describes a 24-year-old female with early-onset breast cancer and a somatic mosaic TP53 PV with a variant allele frequency of 19% in blood, initially missed by panel sequencing. Case 2 concerns a 59-year-old female with multiple primary tumours and two identical TP53 variants detected in two different tissues which initially suggested somatic mosaicism but were consistent with a myelodysplastic syndrome-related clone secondary to homozygous germline ERCC6L2-associated bone marrow failure. These cases highlight the importance of accurately interpreting TP53 variants for correct clinical decision-making. Contextual factors such as age, phenotype, family history and tissue testing must guide diagnosis, treatment and surveillance.

We report a patient with a pathogenic germline variant (PGV) in RB1 and somatic mosaicism for a pathogenic TP53 variant who developed three distinct types of childhood cancer: retinoblastoma, osteosarcoma, and myelodysplastic syndrome (MDS) before the age of 6 years. The patient presented with bilateral retinoblastoma at age 4 weeks and a PGV in RB1 was found. At the very young age of 3 years, she developed an osteosarcoma. A pathogenic variant in TP53 was initially found in bone marrow as part of the diagnostic workup for the patient's third cancer, MDS rapidly progressing to acute myeloid leukemia (AML). Due to the unusual clinical phenotype, cultured fibroblasts from a skin biopsy were examined and identified somatic TP53 mosaicism, with a VAF of 7%. The early debut of osteosarcoma at age 3 years indicates a stronger predisposition than only heritable retinoblastoma would explain, but the development of subsequent MDS could implicate a vulnerability to chemotherapy due to digenic cancer predisposition. This unusual clinical course underlines the need for a better understanding of genetic predisposition to cancer and the interplay between genetic predisposition and treatment effects in optimizing therapeutic strategies and improving long-term outcomes for these patients.

Germline DDX41 variants are found in up to 4% of patients with acute myeloid leukemia (AML) and myelodysplastic neoplasms (MDSs), with a higher prevalence among males and a late onset. In this study, we analyzed 647 Danish patients with suspected myeloid neoplasms to assess the frequency of germline DDX41 variants and the functional impact of variants of uncertain significance (VUSs) using a cell-based assay, CRISPR-Select. We identified 16 DDX41 variants in 30 patients, 14 of whom were confirmed or predicted as germline variants. Eight germline variants were classified as likely pathogenic/pathogenic (LP/P), and three as VUSs. We generated a monoallelic DDX41 K562 cell line and used it for CRISPR-Select assessment of variant effects on proliferation and survival. Pathogenic variants impaired proliferation, while benign variants did not. Missense variants had a more subtle effect than truncating ones, suggesting hypomorphic phenotypes. One DDX41 VUS had a significant negative effect on proliferation, suggesting it to be pathogenic, while the other two had no effect, suggesting them to be benign. These findings confirm a 2.4% frequency of LP/P DDX41 germline variants in Danish patients and demonstrate the value of CRISPR-Select in distinguishing pathogenic from benign variants.

Myeloablative conditioning (MAC) and reduced intensity conditioning (RIC) regimens are both used before allogeneic hematopoietic stem cell transplantation (allo-HCT) for myelofibrosis (MF). The median age of patients with MF treated with allo-HCT is increasing and a high non-relapse mortality (NRM), especially to MAC, has increased utilization of lesser intense non-myeloablative (NMA) regimens. The NMA regimen is used as the standard conditioning regimen before allo-HCT for MF at all transplantation centers in Denmark. We describe the outcomes of a highly homogenously treated, population-derived cohort of patients with MF who received NMA conditioning prior to allo-HCT and identify factors associated to transplantation outcomes. The study is a retrospective cohort study of patients with MF treated with an NMA regimen prior to allo-HCT at Copenhagen University Hospital, Rigshospitalet from 2007 to 2023. Of 70 patients with MF who were treated with allo-HCT for MF from 2007 to 2023, 67 patients received NMA conditioning with fludarabine 90 mg/m2 and total body irradiation of 2 to 4 Gray. These 67 patients had a median age of 61.1 years, 22 patients (33%) had a Karnofsky performance status below 90, and 28 patients (44%) had a hematopoietic stem cell transplantation comorbidity index (HCT-CI) above 2. With a median follow-up time of 3.4 years (range, 0.16-15.58 years), 39 patients (58%) were still alive. Eighteen patients (27%) relapsed and of the 28 patients (42%) who died during the study period, 12 (43%) died from relapse and 16 (57%) from NRM. Median time to neutrophil engraftment, transfusion independency, and platelet engraftment was 21 days (range, 11-119 days), 96 days (range, 0-470 days) and 17 days (range, 0-308 days), respectively, with primary graft failure identified in 13 patients (19.7%). Overall survival (OS) after 1, 3, and 5 years was 77%, 68%, and 61%, respectively, whereas the NRM was 15%, 15%, and 21%, respectively. The cumulative incidence of relapse (CIR) was 24% after 1 year, 28% after 3 years, and 28% after 5 years. Multivariable analysis showed that male sex (hazard ratio (HR) = 5.43, P < .001), graft from unrelated donor (HR = 3.58, P = .018) and HCT-CI above 2 (HR = 2.5, P = .025) remained associated to OS, whereas for progression-free survival, only NRAS mutations remained as an independent factor (HR = 5.88, P = .013). Both male sex (HR = 8.41, P = .037) and graft from unrelated donor (HR = 3.15, P = .043) were associated to NRM in multivariable analysis. Our analysis shows that NMA conditioning in the form of low dose total body irradiation and fludarabine before allo-HCT for MF is feasible. Patients show low 1-year NRM but a relatively high 1-year CIR. Differentiated conditioning with more intensive RIC regiments for younger and fit patients could be considered to reduce the early relapse rate without increasing NRM. In survival analysis, donor-patient relation, patient comorbidity burden and patient sex were independently associated to OS.

PURPOSE:Uveal melanoma (UM) classification systems based on anatomical location show limitations in risk stratification and create ambiguities when tumors span multiple uveal regions. The purpose was to develop a comprehensive staging system for UM integrating tumor volume, clinical factors, and genetic alterations that resolves classification challenges while improving risk stratification. METHODS AND PARTICIPANTS:Nationwide retrospective cohort study of 3696 patients diagnosed with UM in Denmark from 1943 to 2022, including 3062 choroidal melanomas, 245 ciliary body melanomas, and 389 iris melanomas. MAIN OUTCOME MEASURES:Disease-specific survival was analyzed using volume-based tumor categories (T0-T5), clinical risk factors (ciliary body involvement and extraocular extension), and genetic alterations (chromosome 3 and 8q status) to develop an integrated staging system (S0-S6) with genetic enhancement (GS1-GS6). RESULTS:Tumor volume demonstrated strong risk stratification, with T0 tumors (<12 mm3) showing 0% mortality throughout follow-up, whereas 20-year mortality progressively increased from T1 (23%) to T5 (63%). The integrated staging system (S0-S6) showed excellent discrimination with 5-year mortality ranging from 0% (S0) to 54% (S6). Iris melanomas demonstrated distinct survival patterns, with nonring configurations showing minimal mortality (0.5% at 20 years) compared with ring melanomas (21%). Genetic analysis revealed that chromosome aberrations, particularly combined monosomy 3 and 8q-gain (hazard ratio [HR] 12.6, 95% confidence interval [CI], 5.5-29.4), carried stronger prognostic weight than conventional staging parameters, with genetic-enhanced staging providing superior risk stratification (10-year mortality: 0% for GS1 to 77% for GS6). CONCLUSIONS:Tumor volume represents a powerful risk stratifier for UM that can be effectively integrated with clinical risk factors and genetic markers to create a refined staging system. The proposed unified approach incorporating volume-based categorization, clinical factors, and genetic status addresses current classification ambiguities and reflects the distinct natural histories of UM subtypes. A practical scoring sheet is provided for clinical implementation of this staging system. FINANCIAL DISCLOSURE(S):Proprietary or commercial disclosure may be found after the references.

PURPOSE:Cancer predisposition syndromes (CPS) with pediatric onset are the leading known cause of childhood malignancies and are increasingly guiding clinical strategies in pediatric oncology. CPS are placed under evolutionary negative selective pressure, but pediatric pancancer studies have so far failed to investigate genomic evolutionary metrics as a guide to predict penetrance and reveal novel CPS. EXPERIMENTAL DESIGN:Germline whole-genome sequencing was performed in a 5-year prospective, registry-validated, nationwide cohort of individuals diagnosed with cancer before age 18. Evolution-guided burden analysis of private germline variants in constrained genes was compared with 125,748 gnomAD exomes. RESULTS:Across a total of 1,127 participants, 16% carried a pathogenic variant in at least one CPS gene. After genotype-phenotype matching, 9% of children in the prospective cohort (n = 651) carried a variant considered causative, a rate deemed significantly higher than in previous studies [RR, 1.54, 95% confidence interval (CI), 1.37-1.75, P = 1 × 10-14]. As predicted for a disease subject to negative Darwinian selective pressure, compared with reference adults, we found a significant excess of loss-of-function (LoF) variants in the 1,500 most constrained genes (RR, 1.54, 99% CI, 1.21-1.95, P = 4 × 10-6). Surprisingly, this excess was greater than expected, leaving a significant residual enrichment of predicted LoF variants in genes evolutionarily considered the least tolerant to damage (RR, 1.41, 99% CI, 1.09-1.80, P = 4 × 10-4). CONCLUSIONS:The high frequency of LoF variants, including in known CPS, emphasizes the need for systematic and extensive germline genomic mapping as part of the diagnostic workup of patients with childhood cancer and the linkage of such data to disease and response phenotypes to guide future pediatric oncological care and ultimately pave the way for prediagnostic interventional measures.

Whilst biallelic variants in RTEL1 are an established cause of telomere biology disorder (TBD), the significance of heterozygous variants has been more challenging to establish. In this nationwide analysis, we describe 18 individuals with heterozygous pathogenic RTEL1 variants from seven families. All were identified during routine clinical genetic investigation for a variety of indications. Each family carried a different variant in RTEL1. Eight individuals had been diagnosed with a TBD-related disease (five with a hematological disorder, four with pulmonary fibrosis, overlap of one). No cases of clinically significant liver fibrosis had been detected. Cutaneous features of dyskeratosis congenita (abnormal skin pigmentation, oral leukoplakia, nail dysplasia and/or prematurely gray hair) were observed in four individuals. Telomere length (lymphocyte) was measured in nine individuals from six families and was below the 1st percentile in eight individuals. These cases illustrate the wide spectrum of disease and reduced penetrance associated with pathogenic RTEL1 variants, providing a real-world perspective on previous findings from research cohorts. We discuss the challenges surrounding incidental findings, clinical surveillance, and reproductive counseling in this context.

Telomere biology disorder (TBD) can present within a wide spectrum of symptoms ranging from severe congenital malformations to isolated organ dysfunction in adulthood. Diagnosing TBD can be challenging given the substantial variation in symptoms and age of onset across generations. In this report, we present two families, one with a pathogenic variant in ZCCHC8 and another with a novel variant in TERC. In the literature, only one family has previously been reported with a ZCCHC8 variant and TBD symptoms. This family had multiple occurrences of pulmonary fibrosis and one case of bone marrow failure. In this paper, we present a second family with the same ZCCHC8 variant (p.Pro186Leu) and symptoms of TBD including pulmonary fibrosis, hematological disease, and elevated liver enzymes. The suspicion of TBD was confirmed with the measurement of short telomeres in the proband. In another family, we report a novel likely pathogenic variant in TERC. Our comprehensive description encompasses hematological manifestations, as well as pulmonary and hepatic fibrosis. Notably, there are no other reports which associate this variant to disease. The families expand our understanding of the clinical implications and genetic causes of TBD.

Increasing recognition of germline DDX41 variants in patients with hematological malignancies prompted us to provide DDX41-specific recommendations for diagnosis, surveillance, and treatment. Causative germline variants in the DDX41 predispose to the development of myeloid neoplasms (MNs), especially myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Almost 3%-5% of all patients with MDS or AML carry a pathogenic or likely pathogenic germline DDX41 variant, while half of them acquire a somatic second hit in the other allele. DDX41-associated MNs exhibit unique clinical characteristics compared to other hematological malignancies with germline predisposition: MNs occur mostly at advanced age and follow an indolent clinical course. Male carriers are more prone to develop MDS or AML than females. DDX41-associated MN is often hypoplastic, and the malignancy may be preceded by cytopenias.

PURPOSE:To describe the phenotype of Danish patients with genetically verified achromatopsia (ACHM) with special focus on signs of progression on structural or functional parameters, and possible genotype-phenotype correlations. METHODS:Forty-eight patients were identified, with disease-causing variants in five different genes: CNGA3, CNGB3, GNAT2, PDE6C and PDE6H. Longitudinal evaluation was possible for 11 patients and 27 patients participated in a renewed in-depth phenotyping consisting of visual acuity assessment, optical coherence tomography (OCT), fundus autofluorescence, colour vision evaluation, contrast sensitivity, mesopic microperimetry and full-field electroretinography. Foveal morphology was evaluated based on OCT images for all 48 patients using a grading system based on the integrity of the hyperreflective photoreceptor band, the inner segment ellipsoid zone (ISe). Signs of progression were evaluated based on longitudinal data and correlation with age. RESULTS:We found a statistically significant positive correlation between OCT grade and age (Spearman ρ = 0.62, p < 0.0001) and we observed changes in the foveal morphology in 2 of 11 patients with ≥5 years of follow-up. We did not find any convincing correlation between age and functional parameters (visual acuity, retinal sensitivity and contrast sensitivity) nor did we find correlation between structural and functional parameters, or any clear genotype-phenotype correlation. CONCLUSIONS:Some patients with ACHM demonstrate signs of progressive foveal changes in OCT characteristics with increasing age. This is relevant in terms of possible new treatments. However, functional characteristics, such as visual acuity, remained stable despite changing foveal structure. Thus, seen from a patient perspective, ACHM can still be considered a non-progressive condition.