Relapsed Childhood High Hyperdiploid Acute Lymphoblastic Leukemia: Genome-Wide Screening Reveals the Presence of Preleukemic Ancestral Clones and the Secondary Nature of Microdeletions and RTK-RAS Mutations.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2591-2591
Author(s):  
Josef Davidsson ◽  
Kajsa Paulsson ◽  
David Lindgren ◽  
Henrik Lilljebjörn ◽  
Tracy Chaplin ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Structural Changes ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Snp Array ◽  
Prognostic Impact ◽  
Genetic Changes ◽  
Ras Mutations ◽  
Paired Samples

Abstract Abstract 2591 Poster Board II-567 Although childhood high hyperdiploid acute lymphoblastic leukemia is associated with a favorable outcome, 20% relapse. This makes it important to identify these patients already at diagnosis to ensure proper risk-stratification. To identify changes associated with relapse and ascertain the genetic evolution patterns, SNP array and mutation analyses of FLT3, KRAS, NRAS, and PTPN11 were performed on 11 paired diagnostic/relapse samples. The “triples trisomies” +4, +10, and +17 were detected in 64%, a frequency similar to the one generally observed at diagnosis, thus questioning their favorable prognostic impact. Structural changes, mainly cryptic hemizygous deletions, were significantly more common at relapse (P<0.05). No single aberration was linked to relapse, but four deletions, involving IKZF1, PAX5, CDKN2A/B or AK3, were recurrent. Based on the genetic relationship between the paired samples, three groups were delineated: 1) identical genetic changes at diagnosis and relapse (18%), 2) clonal evolution with all changes at diagnosis being present at relapse (18%), and 3) clonal evolution with some changes conserved, lost or gained (64%), suggesting the presence of a preleukemic clone. This ancestral clone was characterized by numerical changes only, with structural changes and RTK-RAS mutations being secondary to the high hyperdiploid pattern and perhaps necessary for overt leukemia. Disclosures: No relevant conflicts of interest to declare.

Outcome of Relapse After Allogeneic Transplantation for Childhood Acute Lymphoblastic Leukemia In Complete Remission. A Study of the Pediatric Diseases and Acute Leukemia Working Parties of the EBMT Group

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1291-1291
Author(s):  
Adriana Balduzzi ◽  
Myriam Labopin ◽  
Vanderson Rocha ◽  
Nabila Elarouci ◽  
Giorgio Dini ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Hematopoietic Cell Transplantation ◽  
Conflicts Of Interest ◽  
Pediatric Population ◽  
Lymphoblastic Leukemia ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Prognostic Impact ◽  
Dismal Prognosis ◽  
B Lineage ◽  
Donor Lymphocyte Infusions

Abstract Abstract 1291 Introduction. Childhood acute lymphoblastic leukemia (ALL) relapse occurring after hematopoietic cell transplantation (HCT) has a very dismal prognosis. Its treatment is still controversial and ranges from palliative treatment or chemotherapy to donor lymphocyte infusions, second transplant or experimental approaches. Objectives. The aim of this study is to assess the actual outcome in a pediatric population. The primary endpoint of this study is the 2-year probability of survival of children with ALL relapsing after allogeneic HCT; the secondary endpoint is the relationship between outcome and time of relapse after transplant, for which the following categories were considered: <3, 3–6, 6–12, > 12 months. Patients. Patients younger than 18 years of age undergoing first HCT from any allogeneic donor for ALL in first (CR1) or second (CR2) remission between January 1st 1998 and December 31st 2007 reported to the EBMT were eligible for the study. Results. Out of 3628 transplanted children with ALL reported to the EBMT, 836 (median age 9 years, male 66%) relapsed at a median of 6 months (range 1–67; 25th, 75th 4, 12 months) after HCT. The HCT was performed in CR1 (60%) or CR2 (40%) for a B-lineage (60%) or T- (13%) or unknown (27%) immunophenotype ALL, from an HLA-matched related (44%), unrelated (59%) or mismatched related (7%) donor, with marrow (61%), peripheral (28%) or umbilical (11%) stem cells. Out of 836, 81% died at a median of 2 months (25th,75th centiles:1,7) and 19% were reported as alive at last follow-up at a median of 22 months after relapse (range: 1–130). The 3-year probability of overall survival (3y-OS) was 14% (SE 1). As to immunophenotype, disease phase and donor type, 3y-OS was 15% (SE 2) in B-lineage and 8% (SE 3) in T-ALL, 18% (SE 2) in patients transplanted in CR1 and 11% (SE 6) in CR2 and 17% (SE 2) in patients transplanted from an HLA-identical sibling and 12% (SE 2) from any other donor. According to time of relapse after transplant, 3-year OS was 6% (SE 2), 10% (SE 2), 15% (SE 2) and 27% (SE 4) in those who relapsed in the first quarter, second quarter, second semester or after the first year, respectively. Donor lymphocyte infusions were reported for 7% and a second HCT for 16% of the 836 relapsed children. The probability of undergoing a second HCT within 1 year after relapse was 17% (SE 1); this probability was 6% for relapses occurring <6 months and 25% for later relapses. 3y-OS of those who underwent a second HCT was 32% (SE 5). Conclusions. The multivariate analyses confirmed the prognostic role of disease phase and immunophenotype, but not of the type of donor, assessed the strong prognostic impact of the time elapsed in CR after HCT before relapse, being earlier relapses at worse outcome compared with later relapses, possibly due to the chance of undergoing a second HCT, which role per se was not statistically significant. Disclosures: No relevant conflicts of interest to declare.

Chromothripsis-Mediated Structural Variations and Clonal Evolution In Recurrent Childhood High Hyperdiploid Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 233-233
Author(s):  
Cai Chen ◽  
Christoph Bartenhagen ◽  
Michael Gombert ◽  
Vera Okpanyi ◽  
Vera Binder ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Copy Number Variations ◽  
Chromosome 3 ◽  
Structural Variations ◽  
Childhood All ◽  
A Genome

Abstract High hyperdiploid acute lymphoblastic leukemia (HeH-ALL) is characterized by 51-67 chromosomes and nonrandom gains of specific chromosomes (X, 4, 6, 10, 14, 17, 18, and 21). It presents the most frequent numerical cytogenetic alteration in childhood pre B-cell ALL occurring in 25-30% of cases. Recurrent disease will affect 15-20%. Pre-leukemic HeH clones are generated in utero, but cooperating oncogenic lesions are necessary for overt leukemia and remain to be determined. Recently, a phenomenon termed chromothripsis has been described in which massive structural variations occur in a single aberrant mitosis. Whole or partial chromosomes are shattered and some fragments are lost in the process of rejoining. Thus, characteristically, chromosomal copy numbers oscillate between two copy number states. Chromothripsis has been suggested to be a tumor-driving alteration that may be present in 2-3% of all human cancers. Its role as a potential cooperating or initiating lesion in HeH-ALL has not been determined. We applied state-of-the-art whole-genome next-generation-sequencing to analyze structural variations in six pediatric patients with recurrent HeH-ALL. Matched sample sets taken at diagnosis, remission and/or relapse were compared. Paired end sequencing was carried out on a Genome Analyzer IIx or a HiSeq 2000 (Illumina), respectively. Reads were aligned against the human reference genome (GRCh37) using BWA. Translocations were detected by GASV. Copy number variations were analyzed by FREEC. Structural variations were validated by PCR/Sanger sequencing and FISH. Of the six patients analyzed, five harbored on average one interchromosomal translocation or intrachromosomal inversion, but one patient presented with massive genomic rearrangements (Figure). These affected chromosome 3, 11, 12 and 20. Ten copy number shifts on chromosome 3 oscillating between two copy number states (2 and 3) indicated that these rearrangements were caused by chromothripsis. Breakpoint sequencing revealed that one of the identified translocations (t(12;20)(p13.1;p12.3)), was indeed a three-loci-rearrangement composed of small fragments derived from chromosomes 3, 12 and 20. Characteristically for chromothripsis, the breakpoints clustered closely. Three breakpoints separated by 224 bp and 64 kb were located in the transducin (beta)-like X-linked receptor 1 (TBL1XR1) gene. Other genes repeatedly targeted included the MACRO domain-containing protein 2 (MACROD2) gene (a deacetylase involved in deacetylation of lysine residues in histones and other proteins), the KIAA1467 gene (a transmembrane protein of the integrin alpha FG-GAP repeat containing 3 (ITFG3) family), and a novel regulatory lincRNA (ENSG00000243276). MACROD2 was previously observed as a target of chromothripsis in a colorectal carcinoma. Thus, the characterized breakpoints may identify fragile genomic sites prone to chromothriptic rearrangement. DNA repair was effectuated by non-homologous-end-joining as typical addition of non-template nucleotides with microhomologies of two to four nucleotides at the breakpoints demonstrated. Copy number profiles of this patient showed that at least two distinct leukemic clones could be identified at diagnosis. One had acquired chromothriptic alterations and presented the dominant clone at relapse indicating chemotherapy resistance and tumor-driving potential. Prior whole-exome sequencing did not reveal mutations in known oncogenes or tumor suppressor genes. Therefore, loss of function or expression of genes affected by chromosomal rearrangements, such as TBL1XR1 that is recurrently mutated in childhood ALL with ETV6-RUNX1 translocation, may account for the tumor-driving effect. All leukemic cells at diagnosis showed conformity concerning number and pattern of whole chromosome gains demonstrating that chromothripsis was not an initiating oncogenic event, but occurred secondary to high hyperdiploidy. Further aberrations (t(4;7), loss of 4q) were gained by the chromothriptic clone and could be detected by FISH in minor subclones pointing at ongoing clonal evolution. Taken together, our study reveals chromothripsis as a novel assisting and tumor-driving lesion in HeH ALL. Chromothripsis in HeH-ALL. Copy number variations and translocations at diagnosis (left) and relapse (right). (magenta: chromothriptic translocations; green: other translocations) Disclosures: No relevant conflicts of interest to declare.

Genomic Heterogeneity In Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1389-1389
Author(s):  
Isaac Yaniv ◽  
Marta Jeison ◽  
Michal Hameiri-Grossman ◽  
Jacque Mardoukh ◽  
Nir Adam Sharon ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Cell Cycle Control ◽  
Snp Array ◽  
Cytogenetic Aberration ◽  
Genome Wide ◽  
Small Cohort ◽  
Balanced Translocations ◽  
A Minor

Abstract Pediatric B cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is characterized by recurrent chromosomal aneuploidies and translocations, which are important for diagnosis and risk stratification. Classical cytogenetic and FISH are routinely used for the diagnosis of most of these alterations, particularly those involving balanced translocations, and numerical changes. Recently, genome wide analysis, such as aCGH and SNP technologies have identified copy number alterations (CNA) in genes involved in lymphoid differentiation: IKZF1, PAX5, EBF1 BTG1; and in cell cycle control: CDNK2A/B, RB1 and ETV6. Multiplex ligation–dependent probe amplification (MLPA) technique has been used for the identification of those focal genetic aberrations in a single assay but it cannot reliably detect aberrations present in less than 50% of cells. Cytoscan HD SNP array (Affymetrix) can overcome this limitation and is the only array which enables independent confirmation of CNA with SNP allelic information. Thus minor genetic subclones missed by other methods, can be detected. The aim of this work was to compare between SNP array and MLPA platforms in a routine diagnostic setting. Samples of 39 pediatric BCP-ALL patients well characterized by classical cytogenetic and FISH at diagnosis, were studied by MLPA P335 (MRCHolland) and SNP array (Affymetrix). In total 273 genomic loci were evaluated, 13 (5%) were not informative, of them 7 were not covered by the MLPA kit and 6 gave not evaluable MLPA results. 231/260 (89%) of the loci showed concordance, 39/260 (15%) with CNA and 192/260 (74%) normal. A total of 68 CNA were detected, 29 were discordant and 39 concordant. IKZF1 deletion was identified by both methods in 5 cases (13%). However, a deletion was identified in 3 additional samples, only by cytoscan. In these cases, the deletion was present in a minor subpopulation (in 45% or less of the cells), while the main cytogenetic aberration was present in the majority of cells (60%-90%). We conclude that although MLPA is a useful technique, it is not sensitive enough to detect small populations. In this small cohort, 3 IKZF1 deletions were missed since they were present only in a minor subclone. Interestingly, one of these patients relapsed, and the relapse clone harbored the IKZF1 deletion. Conclusion Genome-wide sensitive methods identify different subpopulations in each patient, thus detecting previously unrecognized genetic heterogeneities. Disclosures: No relevant conflicts of interest to declare.

Paired Immunophenotype Comparison Of Diagnosis and Relapse Samples In B-Lineage Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4954-4954
Author(s):  
Marion Eveillard ◽  
Richard Garand ◽  
Nelly Robillard ◽  
Soraya Wuilleme ◽  
Caroline Thomas ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Leukemic Cells ◽  
Aberrant Expression ◽  
Blast Cells ◽  
B Lineage

Abstract Although great progress has been made in the treatment of acute lymphoblastic leukemia (ALL), relapse remains a major issue in the follow-up of these patients. Recent data about the emergence of subclones during haematological malignancies suggest that relapses could result from resistant cells initially in minority or from cells driven to resistance by previous treatments. Among the tools allowing for the characterization of leukemic cells, flow cytometry (FCM) is an essential approach. Increasingly used to evaluate minimal residual disease (MRD) based on the immunophenotypic features of the blasts at diagnosis, it can also allow to identify immunophenotypic shifts related to clonal evolution. Such an approach would be best studied by comparing follow-up samples from the same patients. In order to be thorough, this would however require that conditions as similar as possible are applied to both types of cells. This work was designed 1) to compare the immunophenotypic features of B-ALL blast cells with those of normal hematogones, 2)to assess potential immunophenotypic shifts at relapse 3)to determine the stability of markers not classically used at diagnosis during follow-up and their potential utility for MRD. FCM was performed simultaneously on thawed paired samples from 15 patients (9 children aged between 1 and 12 years old and 6 adults aged between 23 and 71 years old) with B-lineage ALL. With a three-tubes 8 colours panel comprising a backbone of CD45, CD34, CD22 and CD10, the expression of 8 markers was examined and compared to that observed on normal hematogones contained in 29 bone marrow samples from healthy donors. These 8 markers were CD7, CD19, CD20, CD24, CD38, CD58, CD81, CD123. Moreover, an additional four colours panel was used to examine the more recently described antigens CD44, CD200, CD304 and Her2Neu. The presence of leukemia associated immunophenotypes (LAIP) was defined as a difference in mean fluorescence intensity (MFI) between hematogones and blasts of at least 2 standard deviations. At diagnosis, the expression of each marker was at variance from that on hematogones yielding LAIP in all patients, with at least 4 aberrant markers (up to 11). Antigens with the most abnormal expression were CD10 (100%), CD24 (93.3%), CD81 (80%), CD38 (60%) and CD58 (53.3%). Antigens with the least aberrant expression were CD19 (20%), CD22 (20%), CD123 (20%), CD34 and CD20 (46,7%). CD44 which is at a low level on hematogones, was present for 80% of the patients at diagnosis and overexpressed in ALL with MLL rearrangement (3/15 cases). CD200 was overexpressed in 73% of the patients while CD304 was present in only 40% of the patients. A single patient was positive for Her2Neu, which remained present at relapse. All patients retained at relapse the same global immunophenotype without any change in the EGIL classification (3 B-I, 8 B-II, 4 B-III) and the difference with hematogones remained. The expression of most markers was similar at diagnosis and relapse. There was no change at all for the expression of CD38 which therefore appears as the most interesting marker for follow-up and MRD in ALL. Only one patient each showed a change in the expression of CD44, CD58 or CD123. As a whole, stable markers were CD58, CD44, CD200, CD81 and CD24 in contrast with CD19, CD22, CD123, CD304, CD24 and CD20 which changed in 27 to 67% of the patients. Four patients displayed no immunophenotypic change at relapse while 3 showed a modification of a single marker. For 5 patients, with respectively 6 and 7 LAIP, two markers were modified at relapse. Three markers changed for the patient with Her2Neu expression. Finally, only two patients (13%) showed major changes possibly associated with the emergence of a new clone. This study confirms that B-ALL blast cells differ immunophenotypically from hematogones, although the latter have been reported to possibly be their normal counterpart. These data moreover comfort the interest of using LAIP in the detection of MRD in multiparameter FCM. Finally, since molecular targets of therapeutic monoclonal antibodies do not shift sensibly, their use can also be considered at relapse. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The genomic landscape of acute lymphoblastic leukemia in children and young adults

Hematology ◽  
2014 ◽  
Vol 2014 (1) ◽  
pp. 174-180 ◽  
Author(s):  
Charles G. Mullighan
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Genetic Basis ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Philadelphia Chromosome ◽  
Chromosome 21 ◽  
Genomic Profiling ◽  
Genetic Changes ◽  
Sequencing Studies

Abstract Our understanding of the genetic basis of childhood acute lymphoblastic leukemia (ALL) has been greatly advanced by genomic profiling and sequencing studies. These efforts have characterized the genetic basis of recently described and poorly understood subtypes of ALL, including early T-cell precursor ALL, Philadelphia chromosome–like (Ph-like) ALL, and ALL with intrachromosomal amplification of chromosome 21, and have identified several rational therapeutic targets in high-risk ALL, notably ABL1-class and JAK-STAT inhibitors in Ph–like ALL. Deep sequencing studies are also refining our understanding of the genetic basis of clonal heterogeneity and relapse. These studies have elucidated the nature of clonal evolution during disease progression and identified genetic changes that confer resistance to specific therapeutic agents, including CREBBP and NT5C2. Genomic profiling has also identified common and rare inherited genetic variants that influence the risk of developing leukemia. These efforts are now being extended to ALL in adolescents and adults with the goal of fully defining the genetic landscape of ALL to further improve treatment outcomes in high-risk populations.

scholarly journals Mutational Landscape, Clonal Evolution Patterns and Role of RAS Mutations in Relapsed Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4068-4068
Author(s):  
Koichi Oshima ◽  
Hossein Khiabanian ◽  
Ana Carolina da Silva Almeida ◽  
Gannie Tzoneva ◽  
Francesco Abate ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Disease Progression ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Wild Type ◽  
Ras Mutations ◽  
Recurrent Mutations ◽  
Glucocorticoid Receptor Gene ◽  
Pediatric All

Abstract Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children. Altogether 90% of pediatric ALL patients achieve a complete hematologic remission with current high dose combination chemotherapy and 80% of them remain leukemia free. However, the outcome for patients showing refractory disease or those whose leukemia relapses after an initial transient response remains disappointingly poor with cure rates of less than 40%. To investigate genetic drivers of relapse and resistance and explore the specific roles of clonal evolution in disease progression and relapse here we performed whole-exome sequence analysis of matched diagnosis, germline (remission) and relapse DNA samples in a panel of 55 pediatric ALL patients including 33 T-cell ALLs and 22 B-cell precursor ALLs. These analyses identified an average of 9 mutations present in diagnostic samples and 17 mutations in relapsed leukemia DNAs. Phylogenetic tree analysis for each of the 48 cases with optimal variant call parameters analyzing their clonal evolution dynamics during disease progression, combined with whole genome sequencing of targeted samples with low exonic mutation input, showed that branched evolution in which relapse clones contain some, but not all genetic lesions present in the major clone at diagnosis as the primary mechanism driving tumor progression and relapse present in 45/48 (94%) cases. In addition, and consistent with previous reports we identified the presence of chemotherapy associated mutations in NT5C2 (10/55), TP53 (3/55), CREBBP (4/55) and the NR3C1 glucocorticoid receptor gene (2/55). However, and most strikingly, 23/27 (85%) recurrently mutated genes in this series with mutations preferentially selected or retained at the time of relapse (mutation never lost in the relapse clone) were not implicated in relapse ALL before (HTR3A, MED12, USP9X, CACNA1H, ODZ3, AACS, SAMD4A, ANO5, PAPPA, NAALADL2, HIST3H2A, FZD7, TBX15, NEB, GREB1L, PLXNA4, SGK223, TSC1, PTPRG, FGF10, SYCP2, TRPM3 and EYS). A branched pattern of genetic evolution and the presence of recurrent mutations selected at relapse support that chemotherapy imposes a strong Darwinian genetic selection in leukemic cell populations. In this context it is worth noting that RAS-MAPK pathway activating mutations in NRAS, KRAS and PTPN11 were present in 24/55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, while in others, RAS mutant clones present at diagnosis were replaced by RAS wild type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Most notably, and in agreement with this hypothesis, inducible expression of mutant KRAS in human ALL lines demonstrate that oncogenic KRAS G12D induces methotrexate resistance, but also improves leukemia response to vincristine; a phenotype perfectly recapitulated in a isogenic ALL leukemia model generated from a conditional inducible Kras G12D knockin mice. Mechanistically, KRAS G12 expression induces MAPK dependent abrogation of methotrexate induced apoptosis. Moreover, Kras mutant tumors show enhanced G2/M cell cycle arrest and apoptosis upon spindle poisoning with vincristine, a phenotype linked with increased PLK phosphorylation and transcriptional down-regulation of mitotic genes. Finally clonal competition assays demonstrate that the differential response to methotrexate and vincristine in isogenic Kras wild type and Kras mutant ALL cells results in clonal dominance of Kras G12D populations in cultures treated with methotrexate, while Kras wild type cells are selected the context of vincristine treatment. In all these results show novel insight on the genetics and mechanisms of clonal selection, disease progression and relapse in ALL and demonstrate a previously unrecognized dual role of RAS mutations in chemotherapy response. Disclosures Loh: Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Influence of the fusion gene ETV6-RUNX1 in the prognosis of the Pediatric B- Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2515-2515
Author(s):  
Alejandro Contento-Gonzalo ◽  
Antonio Jimenez-Velazco ◽  
Alcala-Peña Magdalena ◽  
Manuel Barrios ◽  
Katie Hurst ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Disease Free Survival ◽  
Rank Test ◽  
Prognostic Impact ◽  
Excellent Outcome ◽  
Log Rank Test ◽  
Significant Difference

Abstract Abstract 2515 INTRODUCTION AND OBJECTIVES: The ETV6-RUNX1 (TEL-AML1) rearrangement comes from the translocation of two chromosomes t(12;21)(p12;q22), and represents one of the most frequently detected anomalies (15–30%) in the B-precursor Acute Lymphoblastic Leukemia (B-ALL). It must be identified by polymerase chain reaction (PCR) or fluorescent in situ hybridization (FISH) methods, since this translocation is not detected by conventional cytogenetic techniques. The prognostic value of ETV6-RUNX1 is still a matter of controversy. Recently, the group of the St Jude Children's Hospital reported an excellent outcome in patients carrying the translocation, whereas the BMF group did not find any significant difference in the survival of ETV6-RUNX1 positive patients when compared to ETV6-RUNX1 negative. The aim of our study has been to determine the prognostic impact of the ETV6-RUNX1 rearrangement in patients diagnosed of B-ALL in our Hospital, after a long period of follow-up and with the same Spanish treatment protocols (from PETHEMA and SHOP groups). PATIENTS AND METHODS: All patients with B-ALL diagnosis from January 1997 to May 2011 were included in the study: in total, 114 patients with a mean of age of 6 ys (0.3–14). The type of leukemia was ALL common (83 patients), pro-B (17 patients), pre-B (13 patients) and mature (1 patient). All the children over 1 yr received treatment according to PETHEMA group protocols, adjusted to the risk. Children under 1 yr were treated following SHOP group protocols. Seventeen patients received an allogeneic transplantation. The main clinical features of the positive and negative patients for ETV6-RUNX1 are detailed in table 1. ETV6-RUNX1 assay was performed in our laboratory by RT-PCR, according to the European BIOMED project methodology. RESULTS: ETV6-RUNX1 was found in 31 of the 114 patients (27.2%). These patients showed a significantly higher frequency of myeloid antigens (p<0.001), and were always positive for CD10 (p=0.006). All cases of positive ETV6-RUNX1 were over 2 years old. No significant differences between positive and negative ETV6-RUNX1 were obtained when complete remissions (100 vs 80%), relapse (16 vs 20%) or deaths (10 vs 13%) were analyzed. Furthermore, estimation of disease free survival (DSF) at 14 ys for both groups were similar: 80 ± 8% for positive vs 66 ± 7% for negative (p=0.21, log-rank test). And the same happened for overall survival (OS): 87 ± 7% for positive vs 83 ± 5% for negative (p=0.4, log-rank test). DISCUSSION: In our series, including patients with B-ALL treated with similar protocols with long periods of follow-up, we could not find differences between positive and negative ETV6-RUNX1 patients. It is well known that the intensity of the chemotherapy regimen and the age of inclusion in different protocols may influence the prognosis. Therefore, at present, it is still a matter of discussion if previous reported differences in the B-ALL ETV6-RUNX1 positive group could be explained by a different stratification in risk groups or by different chemotherapy regimens. This work has been funded by a grant from AECC, Carmen Lavigne Prize 2010 Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia

2016 ◽  
Vol 113 (40) ◽  
pp. 11306-11311 ◽  
Author(s):  
Koichi Oshima ◽  
Hossein Khiabanian ◽  
Ana C. da Silva-Almeida ◽  
Gannie Tzoneva ◽  
Francesco Abate ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Mapk Pathway ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Wild Type ◽  
Ras Mutations ◽  
Viral Oncogene ◽  
Mutational Landscape ◽  
Viral Oncogene Homolog

Although multiagent combination chemotherapy is curative in a significant fraction of childhood acute lymphoblastic leukemia (ALL) patients, 20% of cases relapse and most die because of chemorefractory disease. Here we used whole-exome and whole-genome sequencing to analyze the mutational landscape at relapse in pediatric ALL cases. These analyses identified numerous relapse-associated mutated genes intertwined in chemotherapy resistance-related protein complexes. In this context, RAS-MAPK pathway-activating mutations in the neuroblastoma RAS viral oncogene homolog (NRAS), kirsten rat sarcoma viral oncogene homolog (KRAS), and protein tyrosine phosphatase, nonreceptor type 11 (PTPN11) genes were present in 24 of 55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, whereas in others RAS mutant clones present at diagnosis were replaced by RAS wild-type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Consistently, functional dissection of mouse and human wild-type and mutant RAS isogenic leukemia cells demonstrated induction of methotrexate resistance but also improved the response to vincristine in mutant RAS-expressing lymphoblasts. These results highlight the central role of chemotherapy-driven selection as a central mechanism of leukemia clonal evolution in relapsed ALL, and demonstrate a previously unrecognized dual role of RAS mutations as drivers of both sensitivity and resistance to chemotherapy.

scholarly journals Towards Personalized Therapy in Pediatric Acute Lymphoblastic Leukemia; Ras Mutations and Prednisolone Resistance

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 372-372
Author(s):  
Ingrid M. Ariës ◽  
Rosanna van den Dungen ◽  
Marco Koudijs ◽  
Edwin Cuppen ◽  
Emile E. Voest ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Deep Sequencing ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Ion Torrent ◽  
Newly Diagnosed ◽  
Ras Pathway ◽  
Ras Mutations ◽  
Activating Mutations

Abstract Background: The 5-year event-free survival of pediatric precursor-B acute lymphoblastic leukemia (BCP-ALL) has currently reached 80-90%. Targeted drugs are warranted to cure the remaining 10-20%. Prednisolone has been shown a pivotal drug and resistance remains an adverse prognostic factor in BCP-ALL. This study aimed to find new druggable targets that may modulate prednisolone resistance. Methods: Leukemic cells from children with newly diagnosed ALL were isolated from bone marrow aspirates, only samples with ≥ 90% leukemic blasts were used. Protein expression of patient leukemic cells before and after exposure to prednisolone was analyzed by reverse phase protein arrays. Deep sequencing of 26 BCP-ALL patients was performed on the Ion Torrent PGM using the Ion AmpliSeq Cancer Panel. Cytotoxicity of primary patients’ cells to Trametinib, Sorafenib, Crizotinib and AS1517499 in combination with or without prednisolone was determined by the MTT assay. Results: We compared phosphorylation levels of 18 key-signaling proteins in leukemic cells between in vitroprednisolone sensitive and resistant ALL patients. Basal phosphorylation levels of these proteins did not differ between prednisolone resistant and sensitive patients’ cells. Exposure to prednisolone for 48h induced discrepant changes in 7 out of 18 proteins between prednisolone resistant and sensitive cases. Exposure to prednisolone decreased phosphorylation of the RAS-RAF-MEK pathway, STAT6 and c-Met in prednisolone sensitive patients cells but not in resistant patients. These findings prompted us to investigate the presence of RAS-pathway and c-Met activating mutations. We investigated hotspot regions of mutations in the protein coding domains of BRAF, NRAS, HRAS, KRAS, PTPN11, FLT3 and c-Met with Ion Torrent deep sequencing (1000x read depth) in pediatric BCP-ALL patients. We observed that 42% of the BCP-ALL patients tested carried activating NRAS and/or KRAS, FLT3 or PTPN11 mutations, of which 6 cases harbored ≥2 RAS-mutations predominantly associated with presence of multiple minor clones (<30%). Interestingly, we detected more often RAS activating mutations in prednisolone resistant compared to sensitive patients (62% vs. 23%). We next evaluated the prednisolone sensitizing properties of inhibitors directed against MEK1/2 (Trametinib), BRAF (Sorafenib), STAT6 (AS1517499) and cMET (Crizotinib) in primary BCP-ALL patients samples. Remarkably, Trametinib was impressively potent in reversing prednisolone resistance eradicating all leukemic cells of the NRAS/KRAS-mutated BCP-ALL patients. In one NRAS/KRAS-mutated case Sorafenib but not Trametinib sensitized to prednisolone significantly. Conclusion: We observed a relative high frequency of RAS-pathway mutations in children with newly diagnosed BCP-ALL (42%). These mutations were more frequently found in prednisolone resistant cases compared to sensitive cases. Highly resistant leukemic patients’ cells harbouring K/N-RAS mutations were sensitized to prednisolone in the presence of the MEK1/2 inhibitor Trametinib or the BRAF inhibitor Sorafenib. Our data suggest that BCP-ALL patients with RAS-activating mutations may benefit from treatment with Trametinib/Sorafenib and strongly argue to implement these inhibitors in combination with prednisone. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comprehensive Genetic Characterization of Pediatric T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1084-1084
Author(s):  
Kristina Karrman ◽  
Anders Castor ◽  
Mikael Behrendtz ◽  
Erik Forestier ◽  
Linda Olsson ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Large Scale ◽  
Genetic Characterization ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Snp Array ◽  
Cell Receptor ◽  
Consecutive Series ◽  
Cell Acute Lymphoblastic Leukemia

Abstract 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. Disclosures No relevant conflicts of interest to declare.

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