Overview on Aneuploidy in Childhood B-Cell Acute Lymphoblastic Leukemia.

Recent years have brought significant progress in the treatment of B-cell acute lymphoblastic leukemia (ALL). This was influenced by both the improved schemes of conventionally used therapy, as well as the development of new forms of treatment. As a consequence, 5-year survival rates have increased and now exceed 90% in pediatric patients. For this reason, it would seem that everything has already been explored in the context of ALL. However, delving into its pathogenesis at the molecular level shows that there are many variations that still need to be analyzed in more detail. One of them is aneuploidy, which is among the most common genetic changes in B-cell ALL. It includes both hyperdiploidy and hypodiploidy. Knowledge of the genetic background is important already at the time of diagnosis, because the first of these forms of aneuploidy is characterized by a good prognosis, in contrast to the second, which is in favor of an unfavorable course. In our work, we will focus on summarizing the current state of knowledge on aneuploidy, along with an indication of all the consequences that may be correlated with it in the context of the treatment of patients with B-cell ALL.

Commonly Assessed Markers in Childhood BCP-ALL Diagnostic Panels and Their Association with Genetic Aberrations and Outcome Prediction.

Immunophenotypic characterization of leukemic cells with the use of flow cytometry (FC) is a fundamental tool in acute lymphoblastic leukemia (ALL) diagnostics. A variety of genetic aberrations underlie specific B-cell precursor ALL (BCP-ALL) subtypes and their identification is of great importance for risk group stratification. These aberrations include: ETV6::RUNX1 fusion gene, Philadelphia chromosome (BCR::ABL1 fusion gene), rearrangements of the KMT2A, TCF3::PBX1 fusion gene and changes in chromosome number (hyperdiploidy and hypodiploidy). Diagnostic panels for BCP-ALL usually include B-cell lineage specific antigens: CD19, CD10, CD20, maturation stage markers: CD34, CD10, CD38, TdT, IgM and other markers useful for possible genetic subtype indication. Some genetic features of leukemic cells (blasts) are associated with expression of certain antigens. This review comprehensively summarizes all known research data on genotype-immunophenotype correlations in BCP-ALL. In some cases, single molecules are predictive of particular genetic subtypes, i.e., NG2 with KMT2A gene rearrangements or CD123 with hyperdiploidy. However, much more information on possible genotype or prognosis can be obtained with wider (≥8-color) panels. In several studies, a quantitative antigen expression scale and advanced statistical analyses were used to further increase the specificity and sensitivity of genotype/immunophenotype correlation detection. Fast detection of possible genotype/immunophenotype correlations makes multicolor flow cytometry an essential tool for initial leukemia diagnostics and stratification.

Detecting hypodiploidy with endoreduplication and masked hypodiploidy in B-cell acute lymphoblastic leukemia using multicolor flow cytometry.

BACKGROUND: Multicolor flow cytometry-based DNA-ploidy (MFC-ploidy) analysis is a simple, sensitive, and popular method for ploidy analysis in B-cell acute lymphoblastic leukemia (B-ALL). However, the utility of MFC-ploidy in the detection of B-ALL with endoreduplication or masked hypodiploidy has not been reported. Herein, we studied the patterns of MFC-ploidy assessment and its utility to detect B-ALL with hypodiploidy and endoreduplication. METHODS: MFC-ploidy analysis was performed using FxCycle Violet-dye-based method, and cytogenetic ploidy was evaluated using chromosomal-counting and FISH analysis. A total of 20 B-ALL cases with endoreduplication were studied for the patterns of MFC-ploidy analysis and compared with 250 patients with hyperdiploidy and 11 cases with pure hypodiploidy. RESULTS: All B-ALL with endoreduplication revealed two distinct peaks (populations) on MFC-ploidy analysis: the first (hypodiploid) peak (median-DNA-index [DI], 0.82; range, 0.6-0.95) and the second (hyperdiploid) peak with almost twice DI (median-DI, 1.53; range, 1.14-1.75). Cytogenetic findings were available in 19 cases and confirmed hypodiploidy with endoreduplication in 13/19 (68.4%) and only hypodiploidy in 3/19 cases. The remaining three cases showed hyperdiploid blasts in cytogenetic studies. Of these three, two cases had <10% blasts population with hypodiploidy. Thus, masked-hypodiploidy could be diagnosed correctly in 3/19 cases on MFC-ploidy analysis. CONCLUSION: MFC-ploidy analysis shows a characteristic pattern of DNA-ploidy in samples with endoreduplication. It allows the distinction between samples with masked hypodiploidy from true hyperdiploidy. An integrated approach involving cytogenetic and MFC-ploidy detection is very helpful in the risk stratification of B-ALL in routine clinical practice.

A Systematic Cytogenetic Strategy to Identify Masked Hypodiploidy in Precursor B Acute Lymphoblastic Leukemia in Low Resource Settings.

Hypodiploidy with < 40 chromosomes is associated with poor prognosis in B cell precursor acute lymphoblastic leukemia. In some patients, the hypodiploid clone undergoes endoreduplication, resulting in doubling of the number of chromosomes and masquerades as a high hyperdiploid BCP-ALL. Karyotyping reveals metaphases with 50-79 chromosomes masking the hypodiploid clone. Identifying hypodiploidy in such cases requires awareness of non random alterations of chromosomal copy numbers found in hypodiploid BCP-ALL. We used a systematic strategy to identify masked hypodiploidy integrating targeted fluorescence in situ hybridization (FISH) analysis directed towards identifying monosomies of chromosomes 7, 15 and 17 and flow cytometry-based ploidy analysis (FCPA). Of 445 patients diagnosed as BCP ALL, 2.9% (13/445) were classified as hypodiploid including patients with masked hypodiploidy. Karyotype analysis showed hypodiploidy in 3 patients, near triploidy in 4 patients and normal karyotype in 6 patients. Four patients with near triploid clone on karyotype showed either bimodal peak (2 patients) or single low hypodiploid peak (1 patient) or only near triploid peak (1 patient) on FCPA. All 6 patients with normal karyotype revealed either bimodal peak (4 patients) or hypodiploid peak (2 patients) on FCPA. Targeted FISH analysis unmasked hypodiploid clone showing monosomies of chromosomes 7, 15 and 17 in all ten patients. Our algorithm successfully identified masked hypodiploidy in patients, including those with endoreduplication (4 patients) and normal karyotype (6 patients). Integrating FCPA with targeted FISH analysis provides a practical, sensitive and specific approach to identify masked hypodiploidy in low resource settings.

Hypodiploidy in a pediatric patient of T-cell acute lymphoblastic leukemia: a case report.

BACKGROUND: T-cell acute lymphoblastic leukemia is a subtype of acute lymphoblastic leukemia, one of the most common childhood neoplasms. Hypodiploidy is a chromosome abnormality with fewer than 45 chromosomes and is associated with unsatisfactory clinical outcomes in acute lymphoblastic leukemia. CASE PRESENTATION: We report clinical and genetic findings of a 14-year-old male with T-cell acute lymphoblastic leukemia with low-hypodiploidy. The medical history included neck pain for a month, facial nerve palsy on the right side for 6 days, fever, drowsiness, and weakness for 3 days, vomiting, diarrhea for 1 day. The physical examination presented features of hypovolemia, palsy of the facial nerve on the right side, enlarged lymph nodes, hepatosplenomegaly, sore throat, and petechiae of the skin. Radiological images indicated lesions of different organs. Bone marrow biopsy confirmed precursor T-ALL. In the FISH tests, KMT2A and BCR/ABL1 rearrangements were not observed. GTG banding revealed 3 cell clones, which confirmed the hypodiploidy. Multiplex RT-qPCR was performed. STIL/TAL1 (del1p32) gene rearrangement was found in the blast cells. Additional tests were performed using the CytoScan HD microarray technique. Molecular karyotype did not reveal hypodiploidy, but identified other abnormalities such as duplication of chromosomal regions: 4q25q35.2, 6p23.3p11.1 and 8p23.3q24.21, and the loss of heterozygosity of short arm chromosome 9. In two regions of the chromosome biallelic deletions were found at 9p21.3, including the CDKN2A, CDKN2B, IFNA1, MTAP genes and at 10q23.31, containing PTEN. The child died 9 days after diagnosis. CONCLUSIONS: Bone marrow biopsy, GTG banding, FISH techniques, and molecular karyotyping were used to make an accurate diagnosis. This case documents a rapid progression of the disease and unfavorable results of T-cell acute lymphoblastic leukemia with hypodiploidy.

Impact of Novel Targeted Therapies and Cytogenetic Risk Groups on Outcome After Allogeneic Transplantation for Adult ALL.

Novel high-risk groups have recently been identified in adult acute lymphoblastic leukemia (ALL), including Philadelphia-like, therapy-related, and measurable residual disease after induction therapy. Furthermore, modern targeted therapies have recently been incorporated into ALL management; rituximab for CD20-positive and blinatumomab for measurable residual disease after induction therapy or relapsed or refractory disease. Allogeneic hematopoietic cell transplantation (allo-HCT) is recommended as consolidation therapy for high-risk ALL; however, its relative benefit for these high-risk groups and after novel therapies is unclear. We performed an analysis of posttransplantation outcomes in a cohort of 261 consecutive patients who underwent allo-HCT for ALL at the 3-site Mayo Clinic Cancer Center (January 1, 2008-December 31, 2018). With a median (range) follow-up of 22.4 months (0.5-135.0), the 100-day and 5-year cumulative incidences of nonrelapse mortality rates were 6.5% and 26.7%, respectively. The 5-year cumulative incidences of relapse and death were 22.6% and 46.2%, respectively. The 1-year estimate of the composite endpoint of graft-versus-host disease/relapse-free survival was 39.3%. We observed no associations of novel high-risk groups or modern targeted therapies with overall survival, nonrelapse mortality, or relapse in multivariable analysis. An increased risk of relapse was observed with T-ALL (hazard ratio, 2.16; 95% confidence interval, 1.14-4.09; P = .02) and hypodiploidy/near-triploidy (hazard ratio, 2.84; 95% confidence interval, 1.06-7.62; P = .04). Our analysis suggests that novel high-risk groups derive a similar benefit from allo-HCT as traditional high-risk adult ALL and that novel targeted therapies do not seem to independently predict for posttransplantation outcomes. It also calls for further exploration of maintenance strategies after Allo-HCT to prevent relapse in high-risk subgroups.

Near-Haploidy and Low-Hypodiploidy in B-Cell Acute Lymphoblastic Leukemia: When Less Is Too Much.

Hypodiploidy with less than 40 chromosomes is a rare genetic abnormality in B-cell acute lymphoblastic leukemia (B-ALL). This condition can be classified based on modal chromosome number as low-hypodiploidy (30-39 chromosomes) and near-haploidy (24-29 chromosomes), with unique cytogenetic and mutational landscapes. Hypodiploid B-ALL with <40 chromosomes has an extremely poor outcome, with 5-year overall survival rates below 50% and 20% in childhood and adult B-ALL, respectively. Accordingly, this genetic feature represents an adverse prognostic factor in B-ALL and is associated with early relapse and therapy refractoriness. Notably, half of all patients with hypodiploid B-ALL with <40 chromosomes cases ultimately exhibit chromosome doubling of the hypodiploid clone, resulting in clones with 50-78 chromosomes. Doubled clones are often the major clones at diagnosis, leading to "masked hypodiploidy", which is clinically challenging as patients can be erroneously classified as hyperdiploid B-ALL. Here, we summarize the main cytogenetic and molecular features of hypodiploid B-ALL subtypes, and provide a brief overview of the diagnostic methods, standard-of-care treatments and overall clinical outcome. Finally, we discuss molecular mechanisms that may underlie the origin and leukemogenic impact of hypodiploidy and may open new therapeutic avenues to improve survival rates in these patients.

The poor prognosis of low hypodiploidy in adults with B-cell precursor acute lymphoblastic leukaemia is restricted to older adults and elderly patients.

The prognostic significance of low-hypodiploidy has not been extensively evaluated in minimal residual disease (MRD)-oriented protocols for adult acute lymphoblastic leukaemia (ALL). We analysed the outcome of hypodiploid adult ALL patients treated within Programa Español de Tratamientos en Hematología (PETHEMA) protocols. The 5-year cumulative incidence of relapse (CIR) of low-hypodiploid B-cell precursor (BCP)-ALL was significantly higher than that of high-hypodiploids (52% vs. 12%, P = 0.013). Low-hypodiploid BCP-ALL patients aged ≤35 years showed superior survival (71% vs. 21%, P = 0.026) and lower 5-year CIR (17% vs. 66%, P = 0.090) than low-hypodiploids aged >35 years. Older adults and elderly low-hypodiploid BCP-ALL patients show dismal prognosis although achieving an end-induction good MRD response.

Aneuploidy in children with relapsed B-cell precursor acute lymphoblastic leukaemia: clinical importance of detecting a hypodiploid origin of relapse.

Aneuploidy is common in paediatric B-cell precursor acute lymphoblastic leukaemia (ALL). Specific subgroups, such as high hyperdiploidy (>50 chromosomes or DNA Index ≥1·16) and hypodiploidy (<45 chromosomes), predict outcome of patients after primary treatment. Whether aneuploidy has a prognostic value for relapsed disease is yet to be determined. Using DNA index and centromere screening by multiplex ligation-dependent probe amplification, we investigated aneuploidy in 413 children treated for first relapse of B-cell precursor ALL according to the ALL-REZ BFM 2002 protocol. Ten-year event-free survival of patients with high hyperdiploid relapses approached 70%, whereas it was only 40% in low hyperdiploid relapses. Three patients with apparent hyperdiploid relapse had TP53 mutations. In these cases, array-based allelotyping revealed a hypodiploid origin with absence of the hypodiploid founder clone (masked hypodiploidy). Collectively, patients with evident or masked hypodiploid relapses showed an extremely low event-free survival rate of 9%. Importantly, the current relapse risk stratification did not identify cases with masked hypodiploidy as high-risk patients, due to their favourable clinical presentation. In multivariate analysis, hypodiploidy proved to be an independent prognostic factor. This finding supports stratification of relapses with hypodiploid origin into high-risk arms in future trials or allocation of patients to alternative treatment approaches.

TbRRM1 knockdown produces abnormal cell morphology and apoptotic-like death in the bloodstream form of T. brucei.

TbRRM1, an SR-related protein, is involved in transcriptional and post-transcriptional gene expression regulation in procyclic T. brucei. In previous work, we found that TbRRM1 is essential and its depletion leads to cell cycle impairment, aberrant phenotypes and cell loss by apoptotic-like death. Here, we report the findings obtained after TbRRM1 knockdown in bloodstream parasites. Depletion of TbRRM1 in this cell stage led also to growth arrest and cell loss by apoptosis-like death. However, microscopic analysis showed aberrant cell morphology with parasites displaying flagellum detachment and cytokinesis impairment after RNAi induction, suggesting that TbRRM1 could play different roles depending on parasite stage.

Prognostic impact of pretreatment cytogenetics in adult Philadelphia chromosome-negative acute lymphoblastic leukemia in the era of minimal residual disease.

BACKGROUND: The introduction of novel prognostic factors such as minimal residual disease (MRD) and genomic profiling has led to the reevaluation of the role of cytogenetics and other conventional factors in risk stratification for acute lymphoblastic leukemia (ALL). METHODS: This study assessed the impact of baseline cytogenetics on the outcomes of 428 adult patients with Philadelphia chromosome-negative ALL who were receiving frontline chemotherapy. Three hundred thirty patients (77%) were treated with hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone-based regimens, and 98 (23%) were treated with the augmented Berlin-Frankfurt-Munster regimen. RESULTS: The median age was 40 years (range, 13-86 years). One hundred eighty-six patients (43%) had diploid cytogenetics, 32 (7%) had complex cytogenetics (defined as ≥ 5 chromosomal abnormalities), 27 (6%) had low hypodiploidy/near-triploidy (Ho-Tr), 24 (6%) had high hyperdiploidy, and 24 (6%) had a mixed-lineage leukemia (MLL) rearrangement. Patients with an MLL rearrangement, Ho-Tr, or a complex karyotype had significantly worse relapse-free survival (RFS) and overall survival (OS) than the diploid group. According to a multivariate analysis including all the baseline characteristics and MRD status, Ho-Tr and a complex karyotype were independent predictive factors for worse RFS and OS. Furthermore, survival among all cytogenetic groups was similar, regardless of the treatment received. CONCLUSIONS: A complex karyotype and Ho-Tr are adverse prognostic factors for adults with ALL independently of the MRD status. These findings suggest that pretreatment cytogenetics remain a valuable prognostic tool in this population. Cancer 2017;123:459-467. © 2016 American Cancer Society.

Mixed Phenotype Acute Leukemia with Low Hypodiploidy in a Pediatric Patient.

We describe the case of a 16 year-old female with mixed phenotype acute leukemia B/myeloid, NOS (formerly biphenotypic leukemia) with masked hypodiploidy and somatic TP53 and CDKN2A/B deletions. She achieved morphologic remission with lymphoid-directed multi-agent chemotherapy, but experienced an early medullary relapse 11 months from initial diagnosis. Her case details the unusual finding of hypodiploidy in a patient with ambiguous lineage leukemia and highlights the complexity of therapy selection for these high-risk patients.

Genetic and epigenetic characterization of hypodiploid acute lymphoblastic leukemia.

PURPOSE: To investigate the genetic and epigenetic landscape of hypodiploid (<45 chromosomes) acute lymphoblastic leukemia (ALL). METHODS: Single nucleotide polymorphism array, whole exome sequencing, RNA sequencing, and methylation array analyses were performed on eleven hypodiploid ALL cases. RESULTS: In line with previous studies, mutations in IKZF3 and FLT3 were detected in near-haploid (25-30 chromosomes) cases. Low hypodiploidy (31-39 chromosomes) was associated with somatic TP53 mutations. Notably, mutations of this gene were also found in 3/3 high hypodiploid (40-44 chromosomes) cases, suggesting that the mutational patterns are similar in low hypodiploid and high hypodiploid ALL. The high hypodiploid ALLs frequently displayed substantial cell-to-cell variability in chromosomal content, indicative of chromosomal instability; a rare phenomenon in ALL. Gene expression analysis showed that genes on heterodisomic chromosomes were more highly expressed in hypodiploid cases. Cases clustered according to hypodiploid subtype in the unsupervised methylation analyses, but there was no association between chromosomal copy number and methylation levels. A comparison between samples obtained at diagnosis and relapse showed that the relapse did not arise from the major diagnostic clone in 3/4 cases. CONCLUSIONS: Taken together, our data support the conclusion that near-haploid and low hypodiploid ALL are different with regard to mutational profiles and also suggest that ALL cases with high hypodiploidy may harbor chromosomal instability.

Masked hypodiploidy in anaplastic meningiomas by duplication of the original clone found in atypical meningiomas: illustration of the evolution of genetic alterations.

Meningiomas are common, usually benign neoplasms of the central nervous system. Atypical and anaplastic meningiomas can be aggressive, show more rapid growth, and a greater propensity to recur following resection. General consensus believes that genetic abnormalities leading to anaplastic transformation are present at initial tumor presentation; however, this has not been demonstrated by array-comparative genome hybridization. We confirm the hypothesis by showing the evolution of genetic alterations in the transformation of an atypical meningioma to an anaplastic meningioma. Additionally, we provide potential genes responsible for malignant transformation of meningiomas, which, with further research, may provide diagnostic and therapeutic implications.