Luiz Fernando Bazzo Catto

Pathogenic germ line variants causing excessive telomere shortening may result in bone marrow failure, hematopoietic malignancy, and extramedullary complications, such as pulmonary fibrosis, liver cirrhosis, and solid tumors. Patients with short telomeres also develop immunodeficiency with low CD4+ T cells and impaired general immunosurveillance, particularly against solid neoplasms. We investigated a broad spectrum of lymphocyte subsets and myeloid immune cells from human patients with telomere biology disorders (TBDs) and matched healthy volunteers to understand further how the immune system is affected by telomere dysfunction. We used mass cytometry for deep-immunophenotyping peripheral blood mononuclear cells, followed by high-dimensional data analysis. Cytokines, chemokines, and growth factors were assessed in serum. Our results showed profound immune alterations in TBDs beyond those observed in aging, with low naïve lymphocytes and thymic hypofunction. We further observed that T helper (Th) subsets were markedly skewed, with an inverted Th2/Th1 ratio, and low Th17 and Th17.1 levels. T-cell activation and exhaustion markers were upregulated, whereas circulating mucosal-associated invariant T cells were significantly decreased and overactivated. Several serum cytokine levels were positively correlated with telomere length and blood counts, suggesting an association with marrow function. In aggregate, these findings suggest a proinflammatory profile in TBDs. Our data provide new details on how TBD affects immune cells, particularly lymphocytes, which may contribute to the clinical phenotypes.

Immunodeficiency in telomere biology disorders (TBDs) has been described in pediatric patients with severe phenotypes, but is less characterized within the broader TBD spectrum. We collected complete blood counts, lymphocyte subsets, and infection history from 88 consecutive patients with TBD with a median age of 38 years (range, 6-76). Most patients were >18 years old (80/88; 90%) and harbored either a TERT (45%) or TERC germ line mutation (32%). Thirty-two patients (36%) experienced significant infections (opportunistic, recurrent, and/or requiring hospitalization); 47% had lymphopenia, and 3% severe neutropenia. Absolute lymphocyte counts (ALCs) of <0.96 and <1.1 × 103/μL, but not severe neutropenia, were associated with increased infection risk and lower overall survival, respectively. Decreased CD3+ T cells, both CD4+ and CD8+, were associated with bone marrow failure, increased infection risk, and reduced survival. Low CD3+ and CD4+ T cells were associated with solid cancers. Telomere length was shortened across the cohort without correlation with ALC or lymphocyte subsets. In a predominantly adult cohort of TBDs, immunodeficiency was marked by T-cell lymphopenia, possibly a consequence of accelerated aging in the hematopoietic compartment. An ALC cutoff of <1.1 × 103/μL may be a useful biomarker to identify patients with an increased risk of infection, a major cause of death in patients with TBD.

TERRA (telomeric repeat-containing RNA) is a class of long noncoding RNAs transcribed from subtelomeric and telomeric regions. TERRA binds to the subtelomeric and telomeric DNA–forming R-loops (DNA-RNA hybrids), which are involved in telomere maintenance and telomerase function, but the role of TERRA in human cells is not well characterized. Here, we comprehensively investigated for the first time TERRA expression in primary human hematopoietic cells from an exploratory cohort of patients with acute myeloid leukemia (AML), patients with acute lymphoblastic leukemia (ALL), patients with telomere biology disorder (TBD), and healthy subjects. TERRA expression was repressed in primary human hematopoietic cells, including healthy donors, patients with ALL, and patients with TBD, irrespective of their telomere length, except for AML. A second cohort comprising 88 patients with AML showed that TERRA was overexpressed in an AML subgroup also characterized by higher R-loop formation, low TERT and RNAseH2 expression, and a paucity of somatic splicing factor mutations. Telomere length did not correlate with TERRA expression levels. To assess the role of TERRA R-loops in AML, we induced R-loop depletion by increasing RNAseH1 expression in 2 AML cell lines. Decreased TERRA R-loops in AML cell lines resulted in increased chemosensitivity to cytarabine. Our findings indicate that TERRA is uniformly repressed in primary human hematopoietic cells but abnormally expressed in an AML subset with low telomerase.

Carmelo Gurnari, Pedro Henrique Prata, Luiz Fernando B. Catto, Arda Durmaz, Lise Larcher, Marie Sébert, Vincent Allain, Tariq Kewan, Simona Pagliuca, André Luiz

PURPOSE: Secondary myeloid neoplasms (sMNs) remain the most serious long-term complications in patients with aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH). However, sMNs lack specific predictors, dedicated surveillance measures, and early therapeutic interventions. PATIENTS AND METHODS: We studied a multicenter, retrospective cohort of 1,008 patients (median follow-up 8.6 years) with AA and PNH to assess clinical and molecular determinants of clonal evolution. RESULTS: Although none of the patients transplanted upfront (n = 117) developed clonal complications (either sMN or secondary PNH), the 10-year cumulative incidence of sMN in nontransplanted cases was 11.6%. In severe AA, older age at presentation and lack of response to immunosuppressive therapy were independently associated with increased risk of sMN, whereas untreated patients had the highest risk among nonsevere cases. The elapsed time from AA to sMN was 4.5 years. sMN developed in 94 patients. The 5-year overall survival reached 40% and was independently associated with bone marrow blasts at sMN onset. Myelodysplastic syndrome with high-risk phenotypes, del7/7q, and ASXL1, SETBP1, RUNX1, and RAS pathway gene mutations were the most frequent characteristics. Cross-sectional studies of clonal dynamics from baseline to evolution revealed that PIGA/human leukocyte antigen lesions decreased over time, being replaced by clones with myeloid hits. PIGA and BCOR/L1 mutation carriers had a lower risk of sMN progression, whereas myeloid driver lesions marked the group with a higher risk. CONCLUSION: The risk of sMN in AA is associated with disease severity, lack of response to treatment, and patients' age. sMNs display high-risk morphological, karyotypic, and molecular features. The landscape of acquired somatic mutations is complex and incompletely understood and should be considered with caution in medical management.

The choice to postpone treatment while awaiting genetic testing can result in significant delay in definitive therapies in severely pancytopenic patients. Conversely, inherited bone marrow failure (BMF) misdiagnosis can expose patients to ineffectual and expensive therapies, toxic transplant conditioning regimens, and inappropriate use of an affected family member as a stem cell donor. To predict the likelihood of patients having acquired or inherited BMF, we developed a two-step data-driven machine-learning model using 25 clinical and laboratory variables typically recorded at the initial clinical encounter. For model's development, patients were labeled as having acquired or inherited BMF depending on their genomic data. Datasets were unbiasedly clustered and an ensemble model was trained with cases from the largest cluster of the training cohort (n=359) and validated with an independent cohort (n=127). Cluster A, the largest group, was mostly immune or inherited aplastic anemia, whereas Cluster B was composed of underrepresented BMF phenotypes, and not included in the next step of data modeling due to small sample size. The ensemble model Cluster A-specific was accurate (89%) to predict BMF etiology, correctly predicting inherited and likely immune BMF in 79% and 92% of cases, respectively. Our model represents a practical guide for BMF diagnosis and highlights the importance of clinical and laboratory variables in the initial evaluation, particularly telomere length. Our tool can be potentially used by general hematologists and health care providers not specialized in BMF, and in under-resourced centers, to prioritize patients for genetic testing or for expeditious treatment.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematological disorder caused by the expansion of a hematopoietic clone harboring a somatic genetic variant in the PIG-A gene translating into a wide spectrum of clinical and laboratory changes, from intravascular hemolysis, thrombosis, and bone marrow failure to subclinical presentation. In this study, we retrospectively analyzed 87 consecutive cases (39 women; median follow-up, 18 months; range, 0–151 months) in whom a PNH clone was detected by flow cytometry between 2006 and 2019 seen at a single Brazilian referral center. The median age at diagnosis was 29 years (range, 8 to 83 years); 29 patients (33%) were initially classified as PNH/bone marrow failure, 13 (15%) as classic PNH, and 45 (52%) as subclinical PNH. The median overall survival (OS) of the entire cohort was not reached during follow-up, without significant differences between groups. At diagnosis, the median PNH clone size was 2.8% (range, 0 to 65%) in erythrocytes and 5.4% (range, 0 to 80%) in neutrophils. Fourteen patients experienced clone expansion during follow-up; in other 14 patients the clone disappeared, and in 18 patients it remained stable throughout the follow-up. A subclinical PNH clone was detected in three telomeropathy patients at diagnosis, but it was persistent and confirmed by DNA sequencing in only one case. In conclusion, PNH presentation was variable, and most patients had subclinical disease or associated with marrow failure and did not require specific anticomplement therapy. Clone size was stable or even disappeared in most cases.

Summary: Eltrombopag has been added to first‐line treatment of immune aplastic anaemia (AA), resulting in higher responses. We analysed marrow samples of AA patients who responded to immunosuppressive therapy (IST) alone or in combination with eltrombopag for the composition of the haematopoietic stem and progenitor cell (HSPC) compartment. The number of CD34⁺ cells and multipotent progenitors was higher in patients treated with eltrombopag (P < 0·005; P < 0·05; respectively), but not the number of stem cells. No aberrant phenotype was observed. These results indicate that eltrombopag augments CD34⁺ cells in vivo and preferentially expands multipotent progenitors, but not stem cells.

Haploinsufficiency of GATA2 caused by heterozygous loss-of-function mutations is associated with cytopenias and predisposition to myelodysplasia and AML with other variable extrahematopoietic manifestions, including lymphedema, pulmonary alveolar proteinosis, and hearing loss. The authors report on 2 siblings with the disorder whose father was asymptomatic because of an acquired missense mutation in the affected allele that was restricted to hematopoietic cells; surprisingly, he also had no extrahematopoietic complications.