Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.

A comprehensive international consensus on the cytogenetic risk-group stratification of KMT2A-rearranged (KMT2A-r) pediatric acute myeloid leukemia (AML) is lacking. This retrospective (2005-2016) International Berlin-Frankfurt-Münster Study Group study on 1256 children with KMT2A-r AML aims to validate the prognostic value of established recurring KMT2A fusions and additional cytogenetic aberrations (ACAs) and to define additional, recurring KMT2A fusions and ACAs, evaluating their prognostic relevance. Compared with our previous study, 3 additional, recurring KMT2A-r groups were defined: Xq24/KMT2A::SEPT6, 1p32/KMT2A::EPS15, and 17q12/t(11;17)(q23;q12). Across 13 KMT2A-r groups, 5-year event-free survival probabilities varied significantly (21.8%-76.2%; P < .01). ACAs occurred in 46.8% of 1200 patients with complete karyotypes, correlating with inferior overall survival (56.8% vs 67.9%; P < .01). Multivariable analyses confirmed independent associations of 4q21/KMT2A::AFF1, 6q27/KMT2A::AFDN, 10p12/KMT2A::MLLT10, 10p11.2/KMT2A::ABI1, and 19p13.3/KMT2A::MLLT1 with adverse outcomes, but not those of 1q21/KMT2A::MLLT11 and trisomy 19 with favorable and adverse outcomes, respectively. Newly identified ACAs with independent adverse prognoses were monosomy 10, trisomies 1, 6, 16, and X, add(12p), and del(9q). Among patients with 9p22/KMT2A::MLLT3, the independent association of French-American-British–type M5 with favorable outcomes was confirmed, and those of trisomy 6 and measurable residual disease at end of induction with adverse outcomes were identified. We provide evidence to incorporate 5 adverse-risk KMT2A fusions into the cytogenetic risk-group stratification of KMT2A-r pediatric AML, to revise the favorable-risk classification of 1q21/KMT2A::MLLT11 to intermediate risk, and to refine the risk-stratification of 9p22/KMT2A::MLLT3 AML. Future studies should validate the associations between the newly identified ACAs and outcomes and unravel the underlying biological pathogenesis of KMT2A fusions and ACAs.

Genomic characterization of pediatric patients with acute myeloid leukemia (AML) has led to the discovery of somatic mutations with prognostic implications. Although gene-expression profiling can differentiate subsets of pediatric AML, its clinical utility in risk stratification remains limited. Here, we evaluate gene expression, pathogenic somatic mutations, and outcome in a cohort of 435 pediatric patients with a spectrum of pediatric myeloid-related acute leukemias for biological subtype discovery. This analysis revealed 63 patients with varying immunophenotypes that span a T-lineage and myeloid continuum designated as acute myeloid/T-lymphoblastic leukemia (AMTL). Within AMTL, two patient subgroups distinguished by FLT3-ITD and PRC2 mutations have different outcomes, demonstrating the impact of mutational composition on survival. Across the cohort, variability in outcomes of patients within isomutational subsets is influenced by transcriptional identity and the presence of a stem cell–like gene-expression signature. Integration of gene expression and somatic mutations leads to improved risk stratification. Significance: Immunophenotype and somatic mutations play a significant role in treatment approach and risk stratification of acute leukemia. We conducted an integrated genomic analysis of pediatric myeloid malignancies and found that a combination of genetic and transcriptional readouts was superior to immunophenotype and genomic mutations in identifying biological subtypes and predicting outcomes. This article is highlighted in the In This Issue feature, p. 549

One of the most frequent genes affected in pediatric ALL is the CDKN2A/2B gene, acting as a secondary cooperating event and playing an important role in cell-cycle regulation and chemosensitivity. Despite its inclusion in combined CNA (copy-number alterations) classifiers, like the IKZF1plus entity and the UKALL CNA profile, the prognostic impact of the individual gene deletions outside the context of a combined CNA evaluation remains controversial. Addressing the CDKN2A/2B deletions’ additive prognostic effect in current risk-stratification algorithms, we present a retrospective study of a Greek pediatric ALL cohort comprising 247 patients studied over a 24-year period (2000–2023). Herein, we provide insight regarding the correlation with disease features, MRD clearance, and independent prognostic significance for this ALL cohort treated with contemporary BFM-based treatment protocols. Within an extended follow-up time of 135 months, the presence of the CDKN2A/2B deletions (biallelic or monoallelic) was associated with inferior EFS rates (65.1% compared to 91.8% for the gene non-deleted subgroup, p < 0.001), with the relapse rate accounting for 22.2% and 5.9%, respectively (p < 0.001). The presence of the biallelic deletion was associated with the worst outcomes (EFS 57.2% vs. 89.6% in the case of any other status, monoallelic or non-deleted, p < 0.001). Survival differences were demonstrated for B-ALL cases (EFS 65.3% vs. 93.6% for the non-deleted B-ALL subgroup, p < 0.001), but the prognostic effect was not statistically significant within the T-ALL cohort (EFS 64.3 vs. 69.2, p = 0.947). The presence of the CDKN2A/2B deletions clearly correlated with inferior outcomes within all protocol-defined risk groups (standard risk (SR): EFS 66.7% vs. 100%, p < 0.001, intermediate risk (IR): EFS 77.1% vs. 97.9%, p < 0.001, high risk (HR): EFS 42.1% vs. 70.5% p < 0.001 for deleted vs non-deleted cases in each patient risk group); additionally, in this study, the presence of the deletion differentiated prognosis within both MRD-positive and -negative subgroups on days 15 and 33 of induction. In multivariate analysis, the presence of the CDKN2A/2B deletions was the most important prognostic factor for relapse and overall survival, yielding a hazard ratio of 5.2 (95% confidence interval: 2.59–10.41, p < 0.001) and 5.96 (95% confidence interval: 2.97–11.95, p < 0.001), respectively, designating the alteration’s independent prognostic significance in the context of modern risk stratification. The results of our study demonstrate that the presence of the CDKN2A/2B deletions can further stratify all existing risk groups, identifying patient subgroups with different outcomes. The above biallelic deletions could be incorporated into future risk-stratification algorithms, refining MRD-based stratification. In the era of targeted therapies, future prospective controlled clinical trials will further explore the possible use of cyclin-dependent kinase inhibitors (CDKIs) in CDKN2A/2B-affected ALL pediatric subgroups.

Background: Monitoring time trends in the incidence of childhood leukaemias and lymphomas requires efficient and continuous data collecting systems. In countries without official cancer registries, such as Greece, ad hoc nationwide registration of incident childhood leukaemias and lymphomas could help elucidate the underlying aetiology and monitor socioeconomic differentials in health care delivery. Methods: We registered all cases and produced age, gender, type and immunophenotype specific figures and overall crude and age adjusted annual incidence rates and secular trends for 863 leukaemia and 311 lymphoma incident cases diagnosed in children <15 years of age across Greece during 1996–2006, namely the first 11 years of the Nationwide Registry for Childhood Hematological Malignancies. Results: The epidemiological profiles of leukaemias/lymphomas in Greece are similar to those in industrialised countries. No secular trends are observed for either malignancy during the studied period. However, the calculated incidence for leukaemia (46.60 cases per 1 million children annually) is among the highest in the EU-27 (19% higher than average; p<0.001), whereas that for lymphoma (16.8 cases per 1 million children annually) is around the EU-27 average. Conclusions: Minimal secular changes in childhood leukaemias/lymphomas have been noted recently in the EU-27, which cannot be easily explained in countries with small populations. Therefore, centralised EU databases such as the Automated Childhood Cancer Information System (ACCIS) should be enlarged to generate sufficient statistical power for monitoring time trends. It would be interesting to explore whether different lifestyle patterns across the EU might be responsible for the observed excess leukaemia incidence in countries such as Greece.

Antifungal prophylaxis (AFP) is recommended in at-risk hematology-oncology patients. We evaluated the safety of AFP with voriconazole (VRC) in pediatric hematology/oncology patients. A retrospective study of VRC AFP in children with malignancies hospitalized in all 7 Greek pediatric hematology/oncology centers during 2008 to 2012 was conducted. Patients’ demographics, outcome, and adverse event (AE) data were recorded. Four hundred twenty-nine VRC AFP courses in 249 patients (median age 6 y, 55% boys) were studied. The most common underlying diseases were acute lymphoblastic leukemia (51%), non Hodgkin lymphoma (8.6%), and acute myeloid leukemia (7.7%). The median number of VRC courses per patient was 1.7, whereas the median VRC dose was 7 mg/kg (range, 5 to 7 mg/kg) every 12 hours. During the last 2 weeks before AFP, 51% of the patients had received corticosteroids, 43% suffered from severe neutropenia, and 17.3% from mucositis. The median duration of VRC AFP was 17 days (range, 1 to 31 d). A single breakthrough fungemia due to Candida glabrata was recorded. Only 1 patient died due to the underlying disease. The most common AEs reported in 70/429 (16.3%) courses with ≥1 AE were elevated liver enzymes (50%), hypokalemia (24.3%), and ophthalmological disorders (14.3%). The median time of AE onset was 5 days (range, 1 to 21 d). Among 70 AEs reported, 38.5%, 48.4%, and 12.8% were of grade I, II, and III, respectively. VRC prophylaxis in pediatric hematology/oncology patients appears to be well tolerated.