Cryptic TCF3 fusions in childhood leukemia: Detection by RNA sequencing.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood and adolescence. In more than 60% of cases of this heterogeneous disease, a genetic marker is identified via cytogenetic or molecular analyses. TCF3 gene fusions occur in 5%-11% of ALL patients. In < 1%, the TCF3 alteration in ALL leads to a TCF3-HLF fusion gene. Even though this is a very rare event, the detection of a TCF3-HLF fusion gene is associated with a very poor prognosis with incurable relapses in almost all patients. The frequent TCF3-PBX1 fusion gene, which is detectable in 5%-10% of childhood B-cell precursor ALLs and ~3.8% of adult B-cell precursor ALLs, is associated with a rather good prognosis, that is, an observed event-free 5-year survival of approximately 85%. Thus, the distinction of the different partner genes fused to TCF3 is essential for risk assessment. To verify RNA sequencing as a tool for detection of known and unknown fusion genes, we screened 200 cases of pediatric B-cell precursor ALL with "targeted" RNA sequencing in a pilot project in comparison to classical cytogenetic analyses (chromosome R-banding analysis), fluorescence in situ hybridization, and PCR. We observed a TCF3 fusion gene in 6.5% (13/200) of the patients. Ten (5%) patients displayed a TCF3-PBX1 fusion gene, two (1%) patients a TCF3-FLI1 fusion gene, and one (0.5%) patient a TCF3-HLF fusion gene. For the TCF3 fusions, we obtained discrepant results with the different methods, which are described in the article. Taken together, translocations leading to TCF3 fusion genes might appear cryptic and may remain undetected by a single method.

Implementation of RNA sequencing and array CGH in the diagnostic workflow of the AIEOP-BFM ALL 2017 trial on acute lymphoblastic leukemia.

Risk-adapted therapy has significantly contributed to improved survival rates in pediatric acute lymphoblastic leukemia (ALL) and reliable detection of chromosomal aberrations is mandatory for risk group stratification. This study evaluated the applicability of panel-based RNA sequencing and array CGH within the diagnostic workflow of the German study group of the international AIEOP-BFM ALL 2017 trial. In a consecutive cohort of 117 children with B cell precursor (BCP) ALL, array analysis identified twelve cases with an IKZF1plus profile of gene deletions and one case of masked hypodiploidy. Genetic markers BCR-ABL1 (n = 1), ETV6-RUNX1 (n = 25), and rearrangements involving KMT2A (n = 3) or TCF3 (n = 3) were assessed by established conventional techniques such as karyotyping, FISH, and RT-PCR. Comparison of these results with RNA sequencing analysis revealed overall consistency in n=115/117 cases, albeit with one undetected AFF1-KMT2A fusion in RNA sequencing and one undetected ETV6-RUNX1 fusion in conventional analyses. The combined application of RNA sequencing, FISH, and CGH+SNP array reliably detected all genetic markers necessary for risk stratification and will be used as the diagnostic standard workflow for BCP-ALL patients enrolled in the AIEOP-BFM ALL 2017 study. Prospectively, consistent collection of genome-wide CGH+SNP array as well as RNA sequencing data will be a valuable source to elucidate new prognostic lesions beyond established markers of pediatric ALL. In this respect, RNA sequencing identified various gene fusions in up to half of the IKZF1plus (n = 6/12) and B-other (n = 19/36) cases but not in cases with hyperdiploid karyotypes (n = 35). Among these fusions, this study reports several previously undescribed in frame PAX5 fusions, including PAX5-MYO1G and PAX5-NCOA6.

Jumping translocations: Short telomeres or pathogenic TP53 variants as underlying mechanism in acute myeloid leukemia and myelodysplastic syndrome?

Chromosomal rearrangements involving one donor chromosome and two or more recipient chromosomes are called jumping translocations. To date only few cases of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with jumping translocations have been described and the underlying mechanisms remain unclear. Here, we analyzed 11 AML and 5 MDS cases with jumping translocations. The cases were analyzed by karyotyping, FISH, telomere length measurement, and next-generation sequencing with an AML/MDS gene panel. Cases with jumping translocations showed significantly (P < .01) shorter telomeres in comparison to healthy age-matched controls. Additional neo-telomeres were found in two cases. In total, eight cases showed recipient chromosomes with a breakpoint in the centromeric region all of them harboring a pathogenic variant in the TP53 gene (n = 6) and/or a loss of TP53 (n = 5). By contrast, no pathogenic variant or loss of TP53 was identified in the six cases showing recipient chromosomes with a breakpoint in the telomeric region. In conclusion, our results divide the cohort of AML and MDS cases with jumping translocations into two groups: the first group with a telomeric breakpoint of the recipient chromosome is characterized by short telomeres and a possibly telomere-based mechanism of chromosomal instability formation. The second group with a centromeric breakpoint of the recipient chromosome is defined by mutation and/or loss of TP53. We, therefore, assume that both critically short telomeres as well as pathogenic variants of TP53 influence jumping translocation formation.

Pulmonary Transplantation of Human Induced Pluripotent Stem Cell-derived Macrophages Ameliorates Pulmonary Alveolar Proteinosis.

RATIONALE: Although the transplantation of induced pluripotent stem cell (iPSC)-derived cells harbors enormous potential for the treatment of pulmonary diseases, in vivo data demonstrating clear therapeutic benefits of human iPSC-derived cells in lung disease models are missing. OBJECTIVES: We have tested the therapeutic potential of iPSC-derived macrophages in a humanized disease model of hereditary pulmonary alveolar proteinosis (PAP). Hereditary PAP is caused by a genetic defect of the GM-CSF (granulocyte-macrophage colony-stimulating factor) receptor, which leads to disturbed macrophage differentiation and protein/surfactant degradation in the lungs, subsequently resulting in severe respiratory insufficiency. METHODS: Macrophages derived from human iPSCs underwent intrapulmonary transplantation into humanized PAP mice, and engraftment, in vivo differentiation, and therapeutic efficacy of the transplanted cells were analyzed. MEASUREMENTS AND MAIN RESULTS: On intratracheal application, iPSC-derived macrophages engrafted in the lungs of humanized PAP mice. After 2 months, transplanted cells displayed the typical morphology, surface markers, functionality, and transcription profile of primary human alveolar macrophages. Alveolar proteinosis was significantly reduced as demonstrated by diminished protein content and surfactant protein D levels, decreased turbidity of the BAL fluid, and reduced surfactant deposition in the lungs of transplanted mice. CONCLUSIONS: We here demonstrate for the first time that pulmonary transplantation of human iPSC-derived macrophages leads to pulmonary engraftment, their in situ differentiation to an alveolar macrophage phenotype, and a reduction of alveolar proteinosis in a humanized PAP model. To our knowledge, this finding presents the first proof-of-concept for the therapeutic potential of human iPSC-derived cells in a pulmonary disease and may have profound implications beyond the rare disease of PAP.

Routes of Clonal Evolution into Complex Karyotypes in Myelodysplastic Syndrome Patients with 5q Deletion.

Myelodysplastic syndrome (MDS) can easily transform into acute myeloid leukemia (AML), a process which is often associated with clonal evolution and development of complex karyotypes. Deletion of 5q (del(5q)) is the most frequent aberration in complex karyotypes. This prompted us to analyze clonal evolution in MDS patients with del(5q). There were 1684 patients with low and intermediate-risk MDS and del(5q) with or without one additional cytogenetic abnormality, who were investigated cytogenetically in our department, involving standard karyotyping, fluorescence in situ hybridization (FISH) and multicolor FISH. We identified 134 patients (8%) with aspects of clonal evolution. There are two main routes of cytogenetic clonal evolution: a stepwise accumulation of cytogenetic events over time and a catastrophic event, which we defined as the occurrence of two or more aberrations present at the same time, leading to a sudden development of highly complex clones. Of the 134 patients, 61% underwent a stepwise accumulation of events whereas 39% displayed a catastrophic event. Patients with isolated del(5q) showed significantly more often a stepwise accumulation of events rather than a catastrophic event. The most frequent aberrations in the group of stepwise accumulation were trisomy 8 and trisomy 21 which were significantly more frequent in this group compared to the catastrophic event group. In the group with catastrophic events, del(7q)/-7 and del(17p)/-17 were the most common aberrations. A loss of 17p, containing the tumor suppressor gene TP53, was found significantly more frequent in this group compared to the group of stepwise accumulation. This leads to the assumption that the loss of TP53 is the driving force in patients with del(5q) who undergo a sudden catastrophic event and evolve into complex karyotypes.

Comparison of different methods for telomere length measurement in whole blood and blood cell subsets: Recommendations for telomere length measurement in hematological diseases.

Different methods of telomere length measurement are used to identify patients with telomeropathies. In our lab, we established four different methods for telomere length measurement, terminal restriction fragment (TRF) analysis by Southern blot analysis, quantitative PCR (qPCR), quantitative telomere/centromere fluorescence in situ hybridization (T/C-FISH) and fluorescence in situ hybridization combined with flow cytometry (FlowFISH). The methods each have distinct properties and apart from this-according to our experience and data-may have an impact on the individual result. In this study, we therefore compared and validated these methods measuring 154 healthy individuals of different age groups (newborn-81 years). A linear decline was found for every method (Southern blotting 64 bp per year; qPCR 31 bp per year; T/C-FISH 36 bp per year; FlowFISH 50 bp per year). With the equation of the regression line the values of each method (T/S ratio, T/C value, RTL value) can be expressed in absolute kb. All methods showed acceptable accuracy. The analysis indicated good agreement between all methods, with the best agreement between T/C-FISH and FlowFISH. Here, FlowFISH was the most precise, accurate, and reproducible method compared to the other methods. Based on our data, we emphasize the influence of expertise and experience that is required to produce robust and reliable telomere length analyses. Furthermore, we want to provide the scientific community working in diagnostics and research with data-funded advice on how to choose the appropriate method to safely discriminate between natural variability and pathological telomere shortening in individual cases.

Telomere shortening, TP53 mutations and deletions in chronic lymphocytic leukemia result in increased chromosomal instability and breakpoint clustering in heterochromatic regions.

Complex karyotypes are associated with a poor prognosis in chronic lymphocytic leukemia (CLL). Using mFISH, iFISH, and T/C-FISH, we thoroughly characterized 59 CLL patients regarding parameters known to be involved in chromosomal instability: status of the genes ATM and TP53 and telomere length. Interestingly, a deletion of the ATM locus in 11q, independent of the cytogenetic context, was associated with significantly diminished risk (p<0.05) of carrying a mutation in TP53. In patients with loss or mutation of TP53, chromosomal breakage occurred more frequently (p<0.01) in (near-) heterochromatic regions. Median telomere length in patients with complex karyotypes was significantly shorter than that of healthy controls and shorter than in all other cytogenetic cohorts. Furthermore, the median telomere length of patients carrying a TP53 mutation was significantly shorter than without mutation. We conclude that telomere shortening in combination with loss of TP53 induces increased chromosomal instability with preferential involvement of (near-) heterochromatic regions.

Mutational Spectrum of Fanconi Anemia Associated Myeloid Neoplasms.

Individuals with Fanconi anemia (FA) have a high risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), yet the secondary somatic mutations lending to these malignancies remain to be further elucidated. We employed a next-generation sequencing myeloid neoplasia gene panel to determine the mutational spectrum of FA-related MDS/AML. Ten of 16 patients showed missense, nonsense, insertion or duplication mutations in 13 genes. In contrast to findings in MDS in the general population, mutations in genes involved in RNA splicing were rarely affected. Mutations in RUNX1 and genes of the RAS pathway appeared more instrumental in the pathogenesis of FA myeloid malignancies. RUNX1 mutations were associated with more advanced disease. Interestingly, one patient with refractory anemia with ring sideroblasts harbored the SF3B1 p.K700E mutation highlighting the mutation's causative role in MDS with ring sideroblasts even in the context of FA. On the whole, our findings implicate a different genetic architecture of FA MDS/AML from adult sporadic MDS. Notably, the genetic events resemble those described in pediatric MDS.

Identification of a Cryptic Insertion ins(11;X)(q23;q28q12) Resulting in a KMT2A-FLNA Fusion in a 13-Month-Old Child with Acute Myelomonocytic Leukemia.

In pediatric acute myeloid leukemia (AML), chromosomal abnormalities leading to a disruption of the lysine methyltransferase 2A (KMT2A) gene in 11q23 are the most frequent rearrangements. Here, we report on the identification of a novel cryptic insertion, ins(11;X)(q23;q28q12), resulting in a translocation of the KMT2A gene in 11q23, leading to a KMT2A-FLNA fusion in a 13-month-old boy with de novo acute myelomonocytic leukemia, who died 38 days after diagnosis. The patient presented a complex karyotype 48∼49,Y,del(X)(q12),+del(X)(q12),+8,ins(11;X)(q23; q28q12),+19. The identified fusion gene was predicted to be out-of-frame (fusion of portions of KMT2A exon 11 with FLNA exon 11). However, RT-PCR experiments demonstrated that a potentially functional transcript was generated by alternative splicing where KMT2A exon 10 was spliced in-frame to the truncated FLNA exon 11. This case report helps to better understand the rare but potentially severe impact of KMT2A- FLNA fusions in infants with AML to improve prognostic stratification of therapy and clinical management.

A child with Li-Fraumeni syndrome: Modes to inactivate the second allele of TP53 in three different malignancies.

Here we report on a child with Li-Fraumeni syndrome with a de novo TP53 mutation c.818G>A, who developed three malignancies at the age of 4 months, 4 and 5 years, respectively. We show that (i) in the choroid plexus carcinoma, the germline mutation was detected in a homozygous state due to copy-neutral LOH/uniparental disomy, (ii) in the secondary AML, a complex karyotype led to loss of the wild-type TP53 allele, (iii) in the Wilms tumor, the somatic mutation c.814G>A led to compound heterozygosity. The findings show that the complete inactivation of TP53 by different mechanisms is an important step towards tumorigenesis.

Effect of TP53 contact and conformational mutations on cell survival and erythropoiesis of human hematopoietic stem cells in a long term culture model.

TP53 deficiencies characterize myeloid malignancies with a dismal prognosis. To unravel the pathomechanism of TP53 mutations in the development of myeloid malignancies, we analyzed the functional properties of TP53 conformational and contact mutations and TP53 loss in human CD34+ cells. We show for the first time that the analyzed conformational mutations lead to higher cell viability in human hematopoietic stem progenitor cells. In contrast to these conformational mutations, contact mutations interfered with efficient erythropoiesis. These findings show that not only the detection of a TP53 mutation is important, but also the specific mutation may play a role in malignant transformation and progression.

Corrigendum: Clinical and Molecular Heterogeneity of RTEL1 Deficiency.

[This corrects the article on p. 449 in vol. 8, PMID: 28507545.].

Clinical and Molecular Heterogeneity of RTEL1 Deficiency.

Typical features of dyskeratosis congenita (DC) resulting from excessive telomere shortening include bone marrow failure (BMF), mucosal fragility, and pulmonary or liver fibrosis. In more severe cases, immune deficiency and recurring infections can add to disease severity. RTEL1 deficiency has recently been described as a major genetic etiology, but the molecular basis and clinical consequences of RTEL1-associated DC are incompletely characterized. We report our observations in a cohort of six patients: five with novel biallelic RTEL1 mutations p.Trp456Cys, p.Ile425Thr, p.Cys1244ProfsX17, p.Pro884_Gln885ins53X13, and one with novel heterozygous mutation p.Val796AlafsX4. The most unifying features were hypocellular BMF in 6/6 and B-/NK-cell lymphopenia in 5/6 patients. In addition, three patients with homozygous mutations p.Trp456Cys or p.Ile425Thr also suffered from immunodeficiency, cerebellar hypoplasia, and enteropathy, consistent with Hoyeraal-Hreidarsson syndrome. Chromosomal breakage resembling a homologous recombination defect was detected in patient-derived fibroblasts but not in hematopoietic compartment. Notably, in both cellular compartments, differential expression of 1243aa and 1219/1300aa RTEL1 isoforms was observed. In fibroblasts, response to ionizing irradiation and non-homologous end joining were not impaired. Telomeric circles did not accumulate in patient-derived primary cells and lymphoblastoid cell lines, implying alternative pathomechanisms for telomeric loss. Overall, RTEL1-deficient cells exhibited a phenotype of replicative exhaustion, spontaneous apoptosis and senescence. Specifically, CD34+ cells failed to expand in vitro, B-cell development was compromised, and T-cells did not proliferate in long-term culture. Finally, we report on the natural history and outcome of our patients. While two patients died from infections, hematopoietic stem cell transplantation (HSCT) resulted in sustained engraftment in two patients. Whether chemotherapy negatively impacts on the course and onset of other DC-related symptoms remains open at present. Early-onset lung disease occurred in one of our patients after HSCT. In conclusion, RTEL deficiency can show a heterogeneous clinical picture ranging from mild hypocellular BMF with B/NK cell lymphopenia to early-onset, very severe, and rapidly progressing cellular deficiency.

Establishing a murine xenograft-model for long-term analysis of factors inducing chromosomal instability in myelodysplastic syndrome: Pitfalls and successes.

Myelodysplastic syndromes (MDS) are difficult to culture long-term showing the need of a model to study the fate of cells with MDS-abnormalities associated with chromosomal instability (CIN). This approach to establish a xenograft model transplanting human hematopoietic stem cells (HSC) with different independent lentivirally-mediated MDS-related modifications into immunodeficient mice is a long-lasting and tedious experiment with many parameters and every positive as well as non-functioning intermediate step will help the research community. As the establishment of appropriate xenograft models is increasing worldwide we aim to share our experiences to contribute toward minimizing loss of mice and following the "right" approach. Here, modified HSCs were intrafemorally transplanted into NSG and/or NSGS mice: (1) RPS14-haploinsufficiency, (2) TP53-deficiency, (3) TP53 hotspot mutations (R248W, R175H, R273H, R249S). Engraftment was achieved and cytogenetic analyses showed human cells with normal karyotypes. However, in all experiments with NSG mice, mainly control cells or GFP-negative cells were engrafted, not allowing observation of modified HSCs. In NSGS mice, engraftment rate was higher, but mice developed graft-versus-host disease. In summary, engraftment of HSCs is promising and could be used to analyze the induction of CIN. However, the analysis of modified HSCs is limited and further experiments are required to improve this model.

Induction of Chromosomal Instability via Telomere Dysfunction and Epigenetic Alterations in Myeloid Neoplasia.

Chromosomal instability (CIN) is a characteristic feature of cancer. In this review, we concentrate on mechanisms leading to CIN in myeloid neoplasia, i.e., myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The pathogenesis of myeloid neoplasia is complex and involves genetic and epigenetic alterations. Chromosome aberrations define specific subgroups and guide clinical decisions. Genomic instability may play an essential role in leukemogenesis by promoting the accumulation of genetic lesions responsible for clonal evolution. Indeed, disease progression is often driven by clonal evolution into complex karyotypes. Earlier studies have shown an association between telomere shortening and advanced MDS and underlined the important role of dysfunctional telomeres in the development of genetic instability and cancer. Several studies link chromosome rearrangements and aberrant DNA and histone methylation. Genes implicated in epigenetic control, like DNMT3A, ASXL1, EZH2 and TET2, have been discovered to be mutated in MDS. Moreover, gene-specific hypermethylation correlates highly significantly with the risk score according to the International Prognostic Scoring System. In AML, methylation profiling also revealed clustering dependent on the genetic status. Clearly, genetic instability and clonal evolution are driving forces for leukemic transformation. Understanding the mechanisms inducing CIN will be important for prevention and for novel approaches towards therapeutic interventions.