Cytogenetics and genomics in pediatric acute lymphoblastic leukaemia.

The last five decades have witnessed significant improvement in diagnostics, treatment and management of children with acute lymphoblastic leukaemia (ALL). These advancements have become possible through progress in our understanding of the genetic and biological background of ALL, resulting in the introduction of risk-adapted treatment and novel therapeutic targets, e.g., tyrosine kinase inhibitors for BCR::ABL1-positive ALL. Further advances in the taxonomy of ALL and the discovery of new genetic biomarkers and therapeutic targets, as well as the introduction of targeted and immunotherapies into the frontline treatment protocols, may improve management and outcome of children with ALL. In this review we describe the current developments in the (cyto)genetic diagnostics and management of children with ALL, and provide an overview of the most important advances in the genetic classification of ALL. Furthermore, we discuss perspectives resulting from the development of new techniques, including artificial intelligence (AI).

Near-tetraploid T-cell acute lymphoblastic leukaemia in childhood: Results of the AIEOP-BFM ALL studies.

BACKGROUND: Near-tetraploidy-defined by DNA index 1.79-2.28 or 81-103 chromosomes-is a rare cytogenetic abnormality observed both in children and adults with T-cell acute lymphoblastic leukaemia (T-ALL) and its prognostic value is not yet determined. PATIENTS AND METHODS: We report a retrospective study conducted in paediatric patients with newly diagnosed T-ALL treated in AIEOP-BFM ALL 2000 and 2009 studies. 31 near-tetraploid T-ALL patients (1.4%) are compared to T-ALL patients without near-tetraploidy. RESULTS: Near-tetraploid karyotype was associated with lower frequency of high-risk features: white blood cells count at diagnosis ≥100,000/µL (19.3% versus 41.0%, p-value < 0.001), PPR (13.3% versus 35.8%, p-value = 0.01) and minimal residual disease high-risk at the end of consolidation phase Induction B (4.03% versus 14.6%, p-value = 0.001). Complete remission was achieved at the end of induction phase (day 33) in 100% near-tetraploid T-ALL patients, compared to 93.2% T-ALL without near-tetraploidy. CONCLUSION: Overall, we found that near-tetraploid T-ALL in newly diagnosed paediatric patients is associated with low-risk presenting features, with favourable treatment response and outcome.

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.

Transcriptional and Mutational Profiling of B-Other Acute Lymphoblastic Leukemia for Improved Diagnostics.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common cancer in children, and significant progress has been made in diagnostics and the treatment of this disease based on the subtypes of BCP-ALL. However, in a large proportion of cases (B-other), recurrent BCP-ALL-associated genomic alterations remain unidentifiable by current diagnostic procedures. In this study, we performed RNA sequencing and analyzed gene fusions, expression profiles, and mutations in diagnostic samples of 185 children with BCP-ALL. Gene expression clustering showed that a subset of B-other samples partially clusters with some of the known subgroups, particularly DUX4-positive. Mutation analysis coupled with gene expression profiling revealed the presence of distinctive BCP-ALL subgroups, characterized by the presence of mutations in known ALL driver genes, e.g., PAX5 and IKZF1. Moreover, we identified novel fusion partners of lymphoid lineage transcriptional factors ETV6, IKZF1 and PAX5. In addition, we report on low blast count detection thresholds and show that the use of EDTA tubes for sample collection does not have adverse effects on sequencing and downstream analysis. Taken together, our findings demonstrate the applicability of whole-transcriptome sequencing for personalized diagnostics in pediatric ALL, including tentative classification of the B-other cases that are difficult to diagnose using conventional methods.

The Clinical Utility of Optical Genome Mapping for the Assessment of Genomic Aberrations in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most prevalent type of cancer occurring in children. ALL is characterized by structural and numeric genomic aberrations that strongly correlate with prognosis and clinical outcome. Usually, a combination of cyto- and molecular genetic methods (karyotyping, array-CGH, FISH, RT-PCR, RNA-Seq) is needed to identify all aberrations relevant for risk stratification. We investigated the feasibility of optical genome mapping (OGM), a DNA-based method, to detect these aberrations in an all-in-one approach. As proof of principle, twelve pediatric ALL samples were analyzed by OGM, and results were validated by comparing OGM data to results obtained from routine diagnostics. All genomic aberrations including translocations (e.g., dic(9;12)), aneuploidies (e.g., high hyperdiploidy) and copy number variations (e.g., IKZF1, PAX5) known from other techniques were also detected by OGM. Moreover, OGM was superior to well-established techniques for resolution of the more complex structure of a translocation t(12;21) and had a higher sensitivity for detection of copy number alterations. Importantly, a new and unknown gene fusion of JAK2 and NPAT due to a translocation t(9;11) was detected. We demonstrate the feasibility of OGM to detect well-established as well as new putative prognostic markers in an all-in-one approach in ALL. We hope that these limited results will be confirmed with testing of more samples in the future.

BCR-ABL1 positive AML or CML in blast crisis? A pediatric case report with inv(3) and t(9;22) in the initial clone.

The co-occurrence of an inversion inv(3)(q21q26)/GATA2-MECOM and a Philadelphia translocation t(9;22)(q34;q11)/BCR-ABL1 in the context of chronic myeloid leukemia (CML) in blast crisis or acute myeloid leukemia (AML) has only rarely been described. To our knowledge, this co-occurrence has been reported in six pediatric patients with CML but not in pediatric patients with AML. Here, we report on a 7-year-old girl, who, presented with a t(9;22) and inv(3) in 14 of 15 metaphases and an additional monosomy 7 was detected in 5 of these metaphases (ISCN: 46,​XX,​inv(3)(q21q26),​t(9;22)(q34q11)[9]/45,​idem,​-7[5]/46,​XX[1]). The p190 BCR-ABL1 fusion transcript was detected by multiplex PCR and targeted RNA sequencing. Due to these results, a clear distinction between a CML in blast crisis and a BCR-ABL1 positive AML was not possible. The patient was treated according to the treatment recommendations of the AML-BFM study group and additionally received tyrosine kinase inhibitor therapy (Dasatinib). The treatment with Dasatinib was successful in eliminating the inv(3)/t(9;22) clone, but the ancestral inv(3) clone persisted. Based upon these findings we diagnosed an AML with inv(3) and a secondary acquisition of t(9;22). This treatment as well as an allogenic transplantation has led to a complete remission of the disease up to this date (21 months post diagnosis).

Frequency and prognostic impact of PAX5 p.P80R in pediatric acute lymphoblastic leukemia patients treated on an AIEOP-BFM acute lymphoblastic leukemia protocol.

PAX5 is a member of the paired box (PAX) family of transcription factors involved in B-cell development. PAX5P80R has recently been described as a distinct genetic B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) subtype with a favorable prognosis in adults. In contrast, an unfavorable outcome has been observed in children. Our aim was to determine the frequency of PAX5P80R in childhood BCP-ALL treated according to the Associazione Italiana Ematologia ed Oncologia Pediatrica-Berlin-Frankfurt-Muenster (AIEOP-BFM) ALL 2000 protocol and to evaluate its clinical significance within this study cohort. The analyses included 1237 patients with ALL treated in the AIEOP-BFM ALL 2000 trial with complete information for copy number variations (CNVs) of IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, and ERG. A customized TaqMan genotyping assay was used to screen for PAX5P80R . Sanger sequencing was used to confirm PAX5P80R -positive results as well as to screen for second variants in PAX5. Agilent CGH + SNP arrays (e-Array design 85 320; Agilent Technologies) were performed in PAX5P80R -positive patients to verify additional CNVs. Almost 2% (20/1028) of our BCP-ALL cohort were PAX5P80R -positive. White blood cell counts higher than 50 000/µl as well as male sex were significantly (P < .05) associated with PAX5P80R . Most of the PAX5P80R -positive cases were 10 years of age or older. PAX5P80R -positive samples were enriched for deletions affecting PAX5, IKZF1, CDKN2A, and CDKN2B. Compared to PAX5P80R -wildtype BCP-ALL, PAX5P80R -positive patients showed a significantly reduced 5-year overall survival (P = .042). Further studies should evaluate the interaction of PAX5P80R with other genetic aberrations to further stratify intermediate risk pediatric BCP-ALL.

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.

Relapses and treatment-related events contributed equally to poor prognosis in children with ABL-class fusion positive B-cell acute lymphoblastic leukemia treated according to AIEOP-BFM protocols.

ABL-class fusions other than BCR-ABL1 characterize around 2-3% of precursor B-cell acute lymphoblastic leukemia. Case series indicated that patients suffering from these subtypes have a dismal outcome and may benefit from the introduction of tyrosine kinase inhibitors. We analyzed clinical characteristics and outcome of 46 ABL-class fusion positive cases other than BCR-ABL1 treated according to AIEOP-BFM (Associazione Italiana di Ematologia-Oncologia Pediatrica-Berlin-Frankfurt-Münster) ALL 2000 and 2009 protocols; 13 of them received a tyrosine kinase inhibitor (TKI) during different phases of treatment. ABL-class fusion positive cases had a poor early treatment response: minimal residual disease levels of ≥5×10-4 were observed in 71.4% of patients after induction treatment and in 51.2% after consolidation phase. For the entire cohort of 46 cases, the 5-year probability of event-free survival was 49.1+8.9% and that of overall survival 69.6+7.8%; the cumulative incidence of relapse was 25.6+8.2% and treatment-related mortality (TRM) 20.8+6.8%. One out of 13 cases with TKI added to chemotherapy relapsed while eight of 33 cases without TKI treatment suffered from relapse, including six in 17 patients who had not received hematopoietic stem cell transplantation. Stem cell transplantation seems to be effective in preventing relapses (only three relapses in 25 patients), but was associated with a very high TRM (6 patients). These data indicate a major need for an early identification of ABL-class fusion positive acute lymphoblastic leukemia cases and to establish a properly designed, controlled study aimed at investigating the use of TKI, the appropriate chemotherapy backbone and the role of hematopoietic stem cell transplantation. (Registered at: clinicaltrials.gov identifier: NTC00430118, NCT00613457, NCT01117441).

Progressive Immunodeficiency with Gradual Depletion of B and CD4⁺ T Cells in Immunodeficiency, Centromeric Instability and Facial Anomalies Syndrome 2 (ICF2).

Immunodeficiency, centromeric instability and facial anomalies syndrome 2 (ICF2) is a rare autosomal recessive primary immunodeficiency disorder. So far, 27 patients have been reported. Here, we present three siblings with ICF2 due to a homozygous ZBTB24 gene mutation (c.1222 T>G, p. (Cys408Gly)). Immune deficiency in these patients ranged from late-onset combined immunodeficiency (CID) with severe respiratory tract infections and recurrent shingles to asymptomatic selective antibody deficiency. Evident clinical heterogeneity manifested despite a common genetic background, suggesting the pathogenic relevance of epigenetic modification. Immunological follow-up reveals a previously unidentified gradual depletion of B and CD4⁺ T cells in all three presented patients with transition of a common variable immunodeficiency (CVID)-like disease to late-onset-CID in one of them. Considering all previously published cases with ICF2, we identify inadequate antibody responses to vaccines and reduction in CD27⁺ memory B cells as prevalent immunological traits. High mortality among ICF2 patients (20%) together with the progressive course of immunodeficiency suggest that hematopoietic stem cell transplantation (HSCT) should be considered as a treatment option in due time.

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.

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.

The transcriptomes of novel marmoset monkey embryonic stem cell lines reflect distinct genomic features.

Embryonic stem cells (ESCs) are useful for the study of embryonic development. However, since research on naturally conceived human embryos is limited, non-human primate (NHP) embryos and NHP ESCs represent an excellent alternative to the corresponding human entities. Though, ESC lines derived from naturally conceived NHP embryos are still very rare. Here, we report the generation and characterization of four novel ESC lines derived from natural preimplantation embryos of the common marmoset monkey (Callithrix jacchus). For the first time we document derivation of NHP ESCs derived from morula stages. We show that quantitative chromosome-wise transcriptome analyses precisely reflect trisomies present in both morula-derived ESC lines. We also demonstrate that the female ESC lines exhibit different states of X-inactivation which is impressively reflected by the abundance of the lncRNA X inactive-specific transcript (XIST). The novel marmoset ESC lines will promote basic primate embryo and ESC studies as well as preclinical testing of ESC-based regenerative approaches in NHP.

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.