To investigate the genetic and epigenetic landscape of hypodiploid (<45 chromosomes) acute lymphoblastic leukemia (ALL).
Single nucleotide polymorphism array, whole exome sequencing, RNA sequencing, ...and methylation array analyses were performed on eleven hypodiploid ALL cases.
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.
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.
Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease that arises in a multistep fashion through acquisition of several genetic aberrations, subsequently giving ...rise to a malignant, clonal expansion of T-lymphoblasts. The aim of the present study was to identify additional as well as cooperative genetic events in T-ALL.
A population-based pediatric T-ALL series comprising 47 cases was investigated by SNP array and deep sequencing analyses of 75 genes, in order to ascertain pathogenetically pertinent aberrations and to identify cooperative events.
The majority (92%) of cases harbored copy number aberrations/uniparental isodisomies (UPIDs), with a median of three changes (range 0-11) per case. The genes recurrently deleted comprised CDKN2A, CDKN2B, LEF1, PTEN, RBI, and STIL. No case had a whole chromosome UPID; in fact, literature data show that this is a rare phenomenon in T-ALL. However, segmental UPIDs (sUPIDs) were seen in 42% of our cases, with most being sUPID9p that always were associated with homozygous CDKN2A deletions, with a heterozygous deletion occurring prior to the sUPID9p in all instances. Among the 75 genes sequenced, 14 (19%) were mutated in 28 (72%) of 39 analyzed cases. The genes targeted are involved in signaling transduction, epigenetic regulation, and transcription. In some cases, NOTCH1 mutations were seen in minor subclones and lost at relapse; thus, such mutations can be secondary events.
Deep sequencing and SNP array analyses of T-ALL revealed lack of wUPIDs, a high proportion of sUPID9p targeting CDKN2A, NOTCH1 mutations in subclones, and recurrent mutations of genes involved in signaling transduction, epigenetic regulation, and transcription.
Acute lymphoblastic leukemia (ALL) arising in infants less than 1 year of age is characterized by genetic rearrangements of the KMT2A gene (previously MLL) and an exceedingly poor prognosis. We have ...previously shown that infant KMT2A -rearranged (KMT2A -R) ALL has one of the lowest numbers of somatic mutations of any sequenced cancer with a mean of only 1.3 non-silent mutations being present in all leukemia cells per patient (Andersson et al., Nat Genet, 2017). Despite the paucity of mutations, activating mutations within the PI3K/RAS signaling pathway were present in about half of cases, most of which were subclonal with a mutant allele frequency (MAF) <0.30. In addition, some patients harbored multiple activating mutations at varying MAFs, suggesting the presence of multiple low-frequency leukemia clones. Based on these findings, we hypothesized that the genetic landscape of infant KMT2A -R ALL is more clonally heterogeneous than other pediatric leukemias, which might contribute to its poor prognosis as current treatment regimens may not eliminate all clones.
To test our hypothesis, high coverage bulk and single-cell genomics were used to study the mutation profiles of subclones in diagnostic samples from four infants younger than 6 months of age with KMT2A -R ALL. This included patients with t(4;11)(q21;q23) KMT2A-AFF1 (n=3) of which two relapsed and a patient with t(11;19)(q23;p13.3) KMT2A - MLLT1 (n=1) that remains in remission. DNA was isolated from bulk leukemia cells and high-depth paired-end whole-genome sequencing was performed on paired diagnostic and remission samples at an average haploid coverage of 160x and 35x, respectively. In addition, whole-exome sequencing was performed on the diagnostic samples at an average exon coverage of 375x. Somatic alterations, including single-nucleotide variations (SNVs), insertions-deletions (indels), structural variations (SVs), and copy number alterations, were detected using multiple analytical pipelines. In parallel, approximately 75 viable single cells from each diagnostic sample were isolated and the DNA from the cells underwent whole genome amplification using the Fluidigm C1 system.
Analyses of the whole-genome sequencing data from the four infant cases revealed an average of 715 putative sequence variants (range of 54-1199) per case across the genome. Using the combined data from whole-genome- and exome sequencing, an average of 55 (range 51-65) non-silent mutations affecting coding genes or non-coding RNAs were identified, with 83% having support by both sequencing methods, validating their presence. An average of 63% and 92% of the non-silent mutations detected by whole-genome and exome sequencing, respectively, were subclonal, demonstrating the enhanced ability to detect low frequency variants with higher sequencing depth.
Subclonal mutations in genes within the PI3K/RAS signaling pathway were detected in 2/4 cases with each patient harboring both a KRASG12D and a FLT3V491L as well as an additional NRASG13D for one of the cases. The recurrent FLT3V491L mutation is present in COSMIC and has been previously detected in leukemia, but to the best of our knowledge, it has not been experimentally verified to be activating.
We are now performing high-throughput amplicon-based sequencing at the single cells from those patients using 150 targets/cell to determine the number of distinct clones into which the bulk mutations segregate. The targets include a mixture of coding, intergenic variants and SVs. In addition, we are determining if each cell contains a mutation at a selected number of loci that are leukemia-associated mutational hotspots. After acquiring the data, we will reconstruct the clonal architecture of the samples, enabling us to assess the clonal heterogeneity and mutational histories of the leukemias.
Taken together, these data will provide a unique look into the underlying biology of infant KMT2A -R ALL by providing new details about the population genetic diversity and temporal genetic changes that occur during KMT2A -R infant leukemogenesis. These data may also provide insight into the universally poor prognosis of patients with KMT2A -R infant ALL.
No relevant conflicts of interest to declare.
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Introduction. Approximately 3-5% of pediatric ALL patients present with the Philadelphia chromosome (Ph+). In the past, Ph+ ALL was associated with a poor prognosis, with long-term ...event-free-survival (EFS) rates of approximately 30% and most patients allocated to hematopoietic stem cell transplantation (HSCT) in first complete remission (CR1). The COG AALL0031 study showed that continuous high imatinib exposure improved outcome with no increase in toxicity and that HSCT offered no advantage over intensive chemotherapy plus imatinib (Schultz et al, JCO2009). Contemporarily, the EsPhALL 2004-2009 intergroup study of post-induction treatment of Ph+ ALL randomly tested the discontinuous use of imatinib added to BFM ALL high risk chemotherapy after the Induction phase in patients with early response, showing an approximate 10% advantage in long-term DFS. All non-responding patients received imatinib and overall 80% received HSCT in CR1 (Biondi et al, Lancet Oncology 2012). Based on both studies, in 2010 the EsPhALL trial was amended into a single arm study to give all patients, on top of the BFM ALL high risk chemotherapy, imatinib 300/mg/m2/day continuously since day 15 of Induction and to gradually decrease the use of HSCT. We present here the results of this EsPhALL 2010-2014 study.
Methods. Ph+ ALL patients aged 1-17 years were enrolled by 10 national study groups (AIEOP, BFM-G/CH, COALL, FRALLE-EORTC, NOPHO, MRC, DCOG, CPH, PINDA, and HKPHOSG), mainly in Europe. MRD was assessed by PCR using IG/TR targets or BCR/ABL1 in hierarchical order, according to Euro-MRD rules. Eligibility to HSCT depended on early response and, after 2012, on MRD at end of Protocol IB (≥ 5 x 10-4) and at the end of consolidation block 3 (any positivity in patients with MRD< 5 x 10-4 at end of IB). Imatinib was recommended throughout the first year after transplant. Patients who continued on chemotherapy received treatment until 24 months after diagnosis.
Results. Overall 158 patients were registered from January 2010 to December 2014; 3 were not eligible, leaving 155 evaluable patients. Sixty-two patients (40%) were ≥ 10 years, 73 (47%) had ≥50,000 WBC count at diagnosis, 103 (84%) had p190. 102 (66%) patients achieved both early good response and CR1 at end of IA and were classified as good risk, while the remaining 53 (34%) were at poor risk. CR1 was achieved by 151 patients (97%) at the end of Induction and by the remaining 4 at the end of IB. Overall, 59 patients (38%) underwent HSCT in CR1. Nineteen patients discontinued treatment, 17 during chemotherapy, 15 due to serious adverse events (SAE, of which 9 related to imatinib - mainly infections) and the remaining 2 to MRD increase (1) and to transfer to adult unit (1). Among patients who underwent HSCT in CR1, 2 discontinued imatinib after HSCT, due to MRD increase (1) and gastrointestinal toxicity related to imatinib (1). Overall, 185 SAE were observed in 86 (55%) patients. The most frequent SAE was infection which occurred in 50 (32%) patients. Other toxicities included osteonecrosis (7 patients, 5%) and gastrointestinal disorders (9 patients, 6%). On the complete cohort of 155 patients, with a median follow-up of 57 months (range 1-86), the 5-year EFS and survival were 57.0 (SE 4.1) and 71.8 (SE 3.8), respectively. The 5-year cumulative incidence of relapse was 26.9 (SE 3.7). Overall, 65 events occurred, 46 in 96 patients who received chemotherapy only (32 relapses and 14 deaths in CCR) and 19 in 59 HSCT in CR1 (8 relapses and 11 deaths in CCR). Overall, relapses occurred in the BM (22), BM+CNS (11), BM+eye (1) and CNS (6). All deaths in CCR, except for 1 in chemotherapy and 3 after HSCT, were due to infection. Half of the deaths in CCR (12/25) occurred in 2 unexpected clusters, in HSCT performed in 2010-2011 and in chemotherapy-only patients ≥ 10 years. Further investigations did not reveal any common pattern within the clusters.
Conclusions: Addition of imatinib given continuously since day 15 of induction markedly increased CR rate to 97% from 50% of the EsPhALL 2004-2009 study. Moreover, in EsPhALL 2010-2014 only 38% of patients underwent HSCT in CR1 (decreasing from 56% in 2010 to 28% in 2014), compared with 81% in the previous study. In spite of this marked decrease in HSCT, the outcome was similar in the 2 studies: 5-year EFS and survival were 57.0% and 71.8% versus 60.3% and 71.6%, respectively in EsPhALL 2010-2014 versus EsPhALL 2004-2009.
Cazzaniga:Fondazione Tettamanti onlus: Employment; Italian Association for Cancer Research: Research Funding. Saha:Shire: Research Funding. Schrappe:Medac: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding.
A comprehensive genetic characterization comprising conventional chromosome banding, fluorescence in situ hybridization (FISH), and single nucleotide polymorphism (SNP) array analyses as well as ...large-scale sequencing of 75 genes were performed on a consecutive series of 47 pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. An abnormal karyotype was identified in 46% of the cases. Recurrent cytogenetic aberrations comprised T-cell receptor (TCR) translocations and deletions of 6q and 9p. FISH analyses of TCR rearrangements were positive in 26% of the investigated cases. The vast majority (37/39; 95%) of cases analyzed by SNP arrays displayed aberrations, with a median of 3 changes (range 0-11) per case. The genes recurrently deleted were CDKN2A, CDKN2B, LEF1, PTEN, RBI, and STIL. One case displayed chromothripsis involving 6q. No case had a whole chromosome uniparental isodisomy (wUPID); in fact, only one T-ALL of 123 informative cases in the literature has had a wUPID. However, segmental UPIDs (sUPIDs) were seen in 44% of the present cases, with most being sUPID9p. CDKN2A was homozygously deleted in all cases with sUPID9p, with a heterozygous deletion occurring prior to the sUPID9p in all instances. There was no evidence for chromosomal instability when comparing diagnostic and relapse samples. Among the genes sequenced, 14 were mutated in 28 cases. The genes targeted are involved in signaling transduction, epigenetic regulation, and transcription. In some cases, NOTCH1 mutations were seen in minor subclones and lost at relapse, showing that such mutations also can be secondary events. These findings support a multistep leukemogenic process.
No relevant conflicts of interest to declare.
Osteonecrosis (ON) is usually considered treatment related in patients with acute lymphoblastic leukemia (ALL). We report two patients with presentation of ON at the time of ALL diagnosis. Both were ...females and diagnosed with ALL at age 8 and 14 years. In the latter, some symptoms and radiologically verified ON in both knees were still present after the end of ALL therapy. No pediatric patients have previously been reported with ON presenting before initiation of ALL therapy.
Abstract Purpose Improved survival rates of pediatric cancer have drawn attention on how to best facilitate long-term follow up and transition from pediatric to adult care. The transition process is ...multifactorial and necessitates the joint involvement of the patient, the family and the healthcare providers. The purpose of this study was to explore the experiences of support from healthcare services during the transition from adolescence to adulthood described by young adult survivors of pediatric cancer. Methods A mixed method with a convergent parallel design was used to evaluate the experiences of receiving support from healthcare services (eg pediatric oncology and pediatric clinic) during transition from adolescence to adulthood described by young adult survivors of pediatric cancer (n = 213) in a nation wide cross-sectional survey. Results A quantitative assessment of the experienced extent and satisfaction of support from healthcare services to handle physical, mental and social changes to continue life after the disease showed that a majority of the participants had received insufficient support. The qualitative analysis indicated a need for equal roles in healthcare to promote participation, a need to manage and process consequences of the disease, and a need for continuous support. Conclusions During transition to adulthood, there's a need for a personalized care plan that takes a holistic approach towards supporting the young cancer survivor in managing life in the best way. Identifying and handling the individual needs of pediatric cancer survivors is important for providing the resources and support required to increase the likelihood of successful transition to adulthood.
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