Novel Focal Gene Deletions in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia Detected By Array Comparative Genomic Hybridization

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1085-1085
Author(s):  
Ingegerd Ivanov Öfverholm ◽  
Anh Nhi Tran ◽  
Vasilios Zachariadis ◽  
Mats Heyman ◽  
Eva Rudd ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Genetic Markers ◽  
Copy Number ◽  
Array Comparative Genomic Hybridization ◽  
Array Cgh ◽  
Lymphoblastic Leukemia ◽  
Comparative Genomic ◽  
Cell Precursor ◽  
Lymphoid Development

Abstract Cure rates for pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) are now exceeding 80%, however, genetic markers that signal high risk are infrequent and relapses occur across all genetic risk groups. Moreover, a substantial proportion of patients diagnosed with ALL lack risk-associated genetic aberration in their leukemic cells at diagnosis, indicating that additional prognostic markers are needed. Our aim was to better understand the mechanisms underlying leukemic transformation and to identify new genetic markers in ALL; for that purpose we investigated the gene dose landscape in leukemic bone marrow cells from 50 pediatric BCP ALL cases treated according to Nordic Society of Pediatric Hematology and Oncology (NOPHO) protocols. Array comparative genomic hybridization (array-CGH) was performed on DNA isolated from diagnostic bone marrow samples; 30 samples harbored known cytogenetic markers and 20 samples lacked risk-associated cytogenetic markers at diagnosis. The samples were run either on the CytoSure ISCA 4x180K UPD array-CGH platform (Oxford Gene Technology, UK), or an in-house 4x180K custom design array-CGH platform with even genome coverage. In a majority of cases without previously known risk-associated genetic markers we detected copy number alterations (CNAs) recently associated with leukemia development and adverse prognosis, including deletions of the lymphoid development genes IKZF1 and VPREB1. Interestingly, while deletions of IKZF1 consistently occurred together with other CNAs, deletion of VPREB1 was the only somatic imbalance detected in two cases. Deletions of VPREB1 were also present in 55% (5/9) of cases with t(12;21) and associated with a higher white blood cell count in this subtype. Furthermore, deletions of a recently annotated DNA-repair gene, INIP, were recurrent in t(12;21) cases and significantly associated with this subtype (P=0.004). This gene has not previously been described in leukemia. Deletions of the cell-cycle gene RB1 and the hematopoietic signaling gene SH2B3 were present in all cases with intrachromosomal amplification of chromosome 21 (iAMP21, n=3) and both were significantly associated with this subtype (P=0.001 and P<0.001 respectively). The SH2B3 deletion was unique to iAMP21 and has not previously been described as characteristic of this subtype; we are currently investigating this particular subgroup further using high-throughput sequencing technologies and updated results will be presented at the congress. Samples with gain of chromosome 21q (iAMP21 or polysomy 21) showed similarities in the copy number profile, including deletions of the lymphoid development genes BCL11A and BTLA. When we considered the functions of the genes affected by CNAs in the cohort, we found that the majority of samples had concurrent alterations in lymphoid and genome integrity pathways; however, alterations in epigenetic regulators and DNA-repair genes were mutually exclusive in our study. In summary, we detected leukemia-associated as well as novel recurrent deletions and our results indicate that a majority of the cases without risk-associated cytogenetic markers could be assigned to distinct genetic subtypes, and highlights the additional value of array-CGH in the diagnostics of ALL. Disclosures No relevant conflicts of interest to declare.

Genomic DNA Copy Number Alterations Present in AML Bone Marrow Samples with Normal Cytogenetics.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 142-142 ◽  
Author(s):  
Matthew J. Walter ◽  
Rhonda E. Ries ◽  
Jon Armstrong ◽  
Brian O’Gara ◽  
James W. Vardiman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Copy Number ◽  
Genomic Dna ◽  
Array Cgh ◽  
Comparative Genomic ◽  
Copy Number Alterations ◽  
Dna Copy Number ◽  
Prognostic Information ◽  
Normal Cytogenetics ◽  
Copy Number Changes

Abstract Cytogenetics and comparative genomic hybridization (CGH) have been used to identify large genomic amplifications and deletions in all subtypes of acute myeloid leukemia (AML). Up to 15–20% of AML patients have a normal karyotype at diagnosis. While cytogenetic abnormalities confer important prognostic information for patients with AML, there remain differences in the therapeutic response and outcome among patients with the same cytogenetic profile, implying that other, more subtle, genetic abnormalities may exist. We hypothesized that a subset of AML patients with normal cytogenetics may contain genomic DNA copy number changes that are too small to be detected using standard cytogenetic techniques. To address this possibility, we used high-resolution bacterial artificial chromosome (BAC) array CGH technology to examine 31 AML patients with normal cytogenetics. The BAC arrays contain 2,464 BAC clones spotted in triplicate on glass slides, and provide a 1 Mb resolution of the entire human genome. Technical generation of the arrays, hybridization parameters, and analysis were similar to that reported for murine BAC array CGH (Nat Genet. 2001 Dec;29(4):459–64). The 31 AML samples included 4 M0, 8 M1, 10 M2, and 9 M4 patients. Array CGH experiments were performed using 500 nanograms of Cyanine 5 labeled genomic DNA from unmanipulated AML bone marrow, mixed with an equal amount of control DNA (a pool of DNA from 4 cancer-free individuals) labeled with Cyanine 3. Using the human 1 Mb BAC arrays, we identified amplifications and deletions from multiple samples that were confirmed with G-banding cytogenetics [del(7)(q31), del(7)(p11.2), +8, del(11)(q13q23), +21, add(21)(q22), −X, −Y, +Y]. In addition, BAC arrays robustly detected copy number alterations that were identified in as few as 4/21 metaphases. We identified 5/31 (16%) patients with normal cytogenetics that contained altered genomic DNA copy numbers using BAC array CGH. Copy number changes were confirmed for several of these genomic loci using a dye-swap experiment, where the AML DNA was labeled with Cyanine 3, and the control DNA with Cyanine 5. Two of the 5 patients with abnormalities detected using array CGH would be reclassified from “intermediate” to “unfavorable” cytogenetics [del(7)(q31.31q34), add(11)(q23.3qter), and 17(p12pter)]. These results suggest that a subset of AML patients with normal cytogenetics contain genomic copy number alterations that may effect treatment and outcome. Patient # FAB subtype Genomic location Gain or loss Size (Megabase) Dye-Swap confirmed 1 M0 7(q31.31q34) loss 2.0 Not done 1 11(q23.3qter) gain 16.5 Not done 2 M1 11(p14) loss 7.4 Yes 3 M1 11(q13.2q14.1) gain 15.8 Yes 3 19(p) gain 64.0 Yes 4 M2 17(p12pter) gain 8.6 Not Done 5 M2 19(p13.1pter) loss 14.8 Yes 5 12(q13) loss 5.0 Yes

Clinical Utility of Array Comparative Genomic Hybridization for Detection of Chromosomal Abnormalities in Pediatric Acute Lymphoblastic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2275-2275
Author(s):  
Karen Rabin ◽  
Chris Man ◽  
Sharon Plon ◽  
Pulivarthi Rao ◽  
Rizwan Naeem
Keyword(s):  
Comparative Genomic Hybridization ◽  
Clinical Utility ◽  
Array Comparative Genomic Hybridization ◽  
Array Cgh ◽  
Lymphoblastic Leukemia ◽  
Comparative Genomic ◽  
Genomic Hybridization ◽  
Structural Abnormalities ◽  
Pediatric All ◽  
Balanced Translocations

Abstract Chromosomal structural abnormalities in ALL are powerful independent predictors of prognosis, and directly impact choice of therapy. Currently, clinical detection of these abnormalities is based on karyotype and fluorescent in-situ hybridization (FISH), but these methods have limitations. Under optimal circumstances, structural abnormalities are detectable in well over 90% of ALL cases, but in actuality, typical cytogenetic laboratories demonstrate only a 50–60% abnormality detection rate. Karyotype may fail due to unsuccessful cell growth in culture and/or relative overgrowth of normal lymphocytes. FISH is limited by the expense and labor intensity of performing a separate assay for each probe used. Array comparative genomic hybridization (CGH) may have clinical utility as a complementary diagnostic tool in pediatric ALL. Its advantages include the ability to detect copy number changes in regions too small to be identifiable by karyotype; to identify novel abnormalities for which specific FISH probes do not exist in current diagnostic laboratories; and to provide information in as many as 50% of cases which show a failed or normal karyotype. In addition to its clinical utility, array CGH provides a wealth of information which may be mined for discovery of new pathways in leukemogenesis and additional prognostic factors within existing disease subgroups. The main limitation of array CGH is its inability to detected balanced translocations. We evaluated the diagnostic utility of a bacterial artificial chromosome (BAC) array CGH platform, the SpectralChip 2600, with an average resolution of 1.0 MB across the genome. We analyzed 50 pediatric ALL bone marrow specimens obtained at diagnosis, and compared the findings to the clinical results based on karyotype and standard 5-probe FISH panel. The cases ranged from 1–15 years (mean 5 years), with marrow containing between 33–94% leukemic blasts (mean 77%). Each sample was hybridized to the chip with a healthy control of the opposite gender. The sensitivity of array CGH in detecting abnormalities identified by karyotype and FISH was approximately 88%. Several of the abnormalities “missed” by CGH, which lowered the sensitivity score, were subsequently found to be erroneous karyotype calls when followed up with specific FISH probes. In addition, array CGH detected numerous additional areas of amplification and deletion which were subsequently validated by FISH, including in 10 cases for which cytogenetics was either normal or unsuccessful. Loss of 1p31, loss of 7p21, and gain of 16p13 were aberrations that were each noted to occur in three or more different cases, and hence may be worthy of further study. In the future, development of a customized ALL chip which is enriched for probes at sites of known amplification and deletion could further heighten diagnostic sensitivity, obviate the need for performance of multiple FISH tests, and provide valuable information in the substantial number of cases with a normal or failed karyotype analysis. Balanced translocations would still require testing via a multiplex PCR assay or a combination of available FISH probes.

Significance of Copy Number Alterations for Molecular Treatment Response in Childhood Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1434-1434
Author(s):  
Doris Steinemann ◽  
Gunnar Cario ◽  
Martin Stanulla ◽  
Leonid Karawajew ◽  
Marcel Tauscher ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Treatment Response ◽  
Copy Number ◽  
Array Cgh ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Individual Treatment ◽  
Copy Number Alterations ◽  
Childhood All ◽  
Significant Difference

Abstract In vivo response to initial therapy, as assessed by determination of minimal residual disease after five and 12 weeks of treatment, has evolved as one of strong prognostic factors in children with acute lymphoblastic leukemia (ALL) treated according to the BFM regime. It is currently not known if the individual treatment response might be influenced by copy number alterations (CNA) leading to altered gene expression. We compared leukemic genomic profiles of 25 treatment sensitive (MRD-SR) and 25 resistant (MRD-HR) childhood ALL patients by means of high-resolution array-CGH. CNA were found in 46 patients (92%) of both treatment response groups. Microscopic alterations affecting the whole or nearly whole chromosome arm were frequently found, e.g. gain of 21 in 11/50, loss of 9p in 5/50, loss of 8p in 3/50, loss of 20q in 3/50 and loss of 7p in 2/50 or gain of 1q in 2/50. The most significant difference was a gain of chromosome 1q23-qter due to an unbalanced t(1;19), found in 10/25 MRD-SR patients, but in none of the MRD-HR patients (p&lt;0.002). The most frequent CNA in the MRD-HR group were deletions of genomic regions harboring the immunoglobulin genes (Ig), e.g. 2p11.2 in 15 of 25 cases (60%) compared to 7 of 25 in the MRD-SR group (28%) (p=0.045). Combining all Ig loci, significantly more MRD-HR than MRD-SR patients were affected with deletions (17 versus 8 patients, p=0.02). The frequency of other CNA, like loss of 9p21 or gains of 21q, did not differ strongly between the two patient groups. This is the first study evaluating the clinical significance of CNA as detected by array-CGH in childhood ALL and may lead to improved risk classification.

Pax5 Haploinsufficiency Cooperates with BCR-ABL1 to Induce Acute Lymphoblastic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 293-293 ◽  
Author(s):  
ChristoPher B Miller ◽  
Charles G Mullighan ◽  
Xiaoping Su ◽  
Jing Ma ◽  
Michael Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Point Mutation ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Lymphoid Development ◽  
B Lymphoid

Abstract Genes regulating B lymphoid development are somatically mutated in over 40% of B-progenitor acute lymphoblastic leukemia (ALL) cases, with the most common targets being the transcription factors PAX5, IKZF1 (encoding Ikaros), and EBF1. Notably, BCR-ABL1 ALL is characterized by a high frequency of mutations of IKZF1 (85%), PAX5 (55%), and CDKN2A/B (encoding INK4/ARF, 55%), suggesting that these lesions cooperate with BCR-ABL1 in lymphoid leukemogenesis. To examine cooperativity between Pax5 haploinsufficiency and BCR-ABL1, we transplanted Pax5+/+ and Pax5+/− bone marrow cells transduced with MSCV-GFP-IRES-p185 BCR-ABL1 retrovirus into lethally irradiated wild-type C57BL6 recipient mice. Mice transplanted with BCR-ABL1 transduced Pax5+/− marrow developed B progenitor cell ALL with significantly higher penetrance and decreased latency when compared to animals transplanted with BCR-ABL1 transduced Pax5+/+ marrow (median survival 36 vs. 60 days, P=0.0003). The latency of tumor onset was further decreased in the presence of Arf haploinsufficiency (Pax5+/+Arf+/+ 60 days, Pax5+/−Arf+/+ 36 days, Pax5+/−Arf+/− 21 days, P&lt;0.0001). All leukemias were of B cell lineage and were transplantable to secondary recipients. In addition, Southern blot analysis revealed the Pax5+/−Arf+/+ leukemias to be monoclonal, where as the Pax5+/−Arf+/− leukemias were oligoclonal. Importantly, the Pax5+/− leukemias exhibited a more immature B cell immunophenotype than Pax5 wild type leukemias. Moreover, a proportion of the Pax5+/− leukemias (19%) exhibited a very immature early pro B cell immunophenotype (Cd19−, Bp1−), suggesting the possibility of acquired lesions in other key regulators of normal B cell differentiation. To explore this possibility and to identify the total complement of genetic lesions required to generate overt leukemia, we performed genome-wide copy number analysis on 30 murine leukemias (15 Pax5+/+, 15 Pax5+/−) using a custom CGH microarray (Agilent) that interrogated 477,000 autosomal loci, including 18,000 probes covering 20 genes encoding B lymphoid transcription factors and genes targeted by recurring copy number abnormalities (CNAs) in human BCR-ABL1 ALL (Bcl11a, Cdkn2a, Ebf1, Ikzf1, Ikzf2, Ikzf3, Il7r, Lef1, Mdm2, Mef2c, Myb, Pax5, Pten, Rb1, Sfpi1, Sox4, Stat5a, Tcf3, Tcf4, and Trp53). This analysis identified focal recurring CNAs in multiple genes including Cdkn2a/b, Ebf1, Ikzf1, Ikzf2, Ikzf3, and Pax5, each of which is a target of mutation in human B-ALL. Overall, there were on average 3.5 CNAs in Pax5+/+ leukemias versus 0.7 CNAs in Pax5+/− leukemias. Genomic resequencing was also performed on Pax5 and revealed three missense mutations in the DNA binding paired domain (R38H, P80R and G85R), one of which (P80R) is the most common PAX5 point mutation in human B-ALL. All three point mutations are predicted to impair DNA binding of Pax5. Interestingly, the majority of the pro-B cell leukemias that arose in the Pax5+/−Arf+/+ animals were found to harbor mutations (CNAs or point mutation) of the retained Pax5 allele, consistent with the immature immunophenotype. To further explore the relationship between our murine model and human BCR-ABL1 ALL, we performed gene expression profiling of Pax5+/+ and Pax5+/− leukemias and compared their signatures to those of human BCR-ABL1 ALL and stage-specific murine B lymphoid developmental signatures using gene set enrichment analysis (GSEA). This analysis identified significant similarity between murine and human BCR-ABL1 leukemias, thus providing further evidence that this model closely recapitulates human BCR-ABL1 ALL. Notably, Pax5+/− leukemias, or Pax5+/+ leukemias that acquired additional mutations of B-lymphoid regulators exhibited a less mature gene expression profile than leukemias lacking B-lymphoid regulatory mutations. These data indicate that loss of Pax5 contributes to leukemogenesis, that additional genomic alterations in genes regulating B lymphoid development and cell cycle regulators/tumor suppressors (Arf) are frequent events in BCR-ABL1 acute lymphoblastic leukemia, and that these lesions result in impaired B-lymphoid maturation in B-ALL. The genetic complexity of BCR-ABL1 ALL is likely to have important therapeutic implications for this poor prognosis subtype of leukemia.

Amplification of RUNX1 gene in two new cases of childhood B-cell precursor acute lymphoblastic leukemia: A case report

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e21000-e21000
Author(s):  
A. Fauzdar ◽  
A. Mahajan ◽  
D. Jain ◽  
M. Mishra ◽  
V. Raina
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Chromosome 21 ◽  
Fish Analysis ◽  
Cell Precursor ◽  
Childhood All ◽  
Mll Gene ◽  
Runx1 Gene

e21000 Background: Chromosome abnormalities of leukemia cells have important prognostic significance in childhood acute lymphoblastic leukemia (ALL). B-cell precursor acute lymphoblastic leukemia (BCP-ALL) ETV6/RUNX1 (alias TEL/AML1) is most frequent i.e. 15 - 35% in the children with 2 - 18 age group. We report two new cases with Pre B- cell ALL without ETV6/RUNX1 rearrangement, showing amplification of AML1 gene detected by FISH analysis. Methods: Bone marrow samples were analyzed for chromosomal abnormalities with conventional G-banding techniques and interphase fluorescence in situ hybridization (FISH) using probes to detect BCR/ABL t(9;22)(q34-q11) fusion, cryptic TEL/AML1 t(12:21)(p13-q22) and MLL rearrangement for del 11q23. Results: In first case a 3-year girl with four copies of AML (RUNX1) gene were observed in 95% of the cell with normal two copies of TEL (ETV6) gene in both interphase and metaphase FISH. We observed BCR-ABL negative translocation and no MLL gene rearrangement in all the interphase cells after doing FISH. We got a normal 46XX karyotype from bone marrow with conventional cytogenetics (CC) in the same patient. In second case, a 4-year male we observed four copies of AML and two copies of TEL gene in more than 80% of cells. In this patient, we got BCR-ABL negative translocation and three copies of MLL gene without any rearrangement through FISH. We got normal 46XY karyotype in the same patient through CC. Conclusions: In both the patients, we observed hyperdiploidy detected with four copies of RUNX1 gene showing tetrasomy of chromosome 21 detected with metaphase FISH analysis whereas G-banding shows normal diploidy. Bone marrow karyotype in combination with molecular cytogenetic techniques like FISH should be done for improvement in sensitivity and accurate cytogenetic analysis in childhood ALL patients for proper identification of prognostic group for optimum treatment. This is one of the few reported studies worldwide for amplification of RUNX1 gene from Indian subcontinent in childhood BCP-ALL. No significant financial relationships to disclose.

Genomic Profiling helps to Predict Treatment Response and Outcome in Relapsed Childhood ETV6/RUNX1-Positive Acute Lymphoblastic Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1485-1485
Author(s):  
Almut Giese ◽  
Reinhard Ullmann ◽  
Cornelia Eckert ◽  
Renate Kirschner-Schwabe ◽  
Guenter Henze ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
High Resolution ◽  
X Chromosome ◽  
Copy Number ◽  
Array Cgh ◽  
Lymphoblastic Leukemia ◽  
Response To Treatment ◽  
Genomic Profiling ◽  
Copy Number Alterations ◽  
Genetic Aberrations

Abstract The ETV6/RUNX1 fusion, resulting from the cryptic translocation t(12;21)(p13;q22), is the most common genetic rearrangement in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) with a prevalence of approximately 20–25% at first presentation. Although generally associated with favorable risk features and advantageous long-term survival rates, similar frequencies of ETV6/RUNX1 positivity at relapse question its prognostic significance. At relapse, a second continuous complete remission can again be achieved in the majority of patients with ETV6/RUNX1-positive ALL, however, a substantial proportion (approximately 25%) exhibit a poor response to treatment and eventually experience a subsequent relapse. Currently, critical secondary genetic events are assumed to be pivotal for t(12;21) positive ALL leukemogenesis. At the chromosomal level, additional numeric aberrations may also contribute to differences both in treatment response and course of disease, and may provide additional prognostic information. Aiming at identifying the incidence and the clinical relevance of additional genetic aberrations at the time point of first ALL relapse diagnosis, we performed whole genome high resolution tiling-path bacterial artificial chromosome (BAC) array CGH of leukemic cell DNA from 53 patients with ETV6/RUNX1-positive first relapse enrolled in the relapse trials ALL-REZ BFM of the Berlin-Frankfurt-Münster study group. Additional genetic aberrations were detected in all of the ETV6/RUNX1-positive leukemic bone marrow samples with a mean number of seven aberrations per ALL. Chromosomal losses were observed approximately 2.5 times more frequently than gains. A high proportion of the identified copy number alterations occurred in recurrently affected chromosomal regions. Copy number alterations most commonly detected by high-resolution array CGH were losses of 12p13 (49%, 26/53), 6q21 (34%, 18/53), 15q15.1 (23%, 12/53), 9p21 (21%, 11/53), 3p21 (21%, 11/53), 5q (19%, 10/53), 19q13 (17%, 9/53), 10q (13%, 7/53), 3p14.2 (11%, 6/53) and gains of 21q22 (32%, 17/53) and of 12p (21%, 11/53). Loss of the whole X-chromosome, detected in 17% (9/53) of the analyzed samples, was observed exclusively in females. In contrast, gain of Xq, identified in 13% (7/53) of the samples, was detectable solely in males. Evaluation of the clinical parameters in relation to recurrent copy number alterations revealed that loss of the whole X-chromosome was associated with a molecular good response to treatment (P=0.03), as assessed by sensitive minimal residual disease (MRD) monitoring. In contrast, loss of 5q31.3, detected in 11% (6/53) of the analyzed samples, was associated with a dismal molecular response to therapy (P=0.019) and with a tendency to a worse outcome (P=0.054). Thus, this study demonstrates that relapsed ETV6/RUNX1-positive ALL is characterized by (a.) multiple additional genomic alterations, in particular by a high incidence of chromosomal losses, which occur predominantly in recurrently affected chromosomal regions, some of which (b.) are of significant prognostic relevance. These findings support the notion that several additional chromosomal changes are not only required for the process of malignant transformation in ETV6/RUNX1-positive ALL (leukemogenesis) but also influence therapeutic success. Perspectively, high resolution genomic profiling will provide valuable information enabling a more refined, individualized therapy and an improved risk stratification in relapsed childhood ALL.

Heterogeneity Of CXCR4 Expression In Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4952-4952
Author(s):  
Tomasz Szczepanski ◽  
Urszula Malek ◽  
Lukasz Sedek ◽  
Alicja Sonsala ◽  
Joanna Zawitkowska ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Residual Disease ◽  
Risk Group ◽  
Lymphoblastic Leukemia ◽  
Cxcr4 Expression ◽  
Study Group ◽  
Cell Precursor ◽  
Minimal Residual

Abstract Background CXCR4 (CD184) is a receptor specific to the Stromal Derived Factor 1 (SDF-1), a ligand also known as CXCL12. The ligand-receptor interaction has a pleiotropic effect on hematopoietic cell proliferation, migration and activation through several signaling pathways. CXCR4 expression on neoplastic cells might be responsible for their dissemination to particular organs with cells expressing CXCL12 (e.g. lymph nodes, bones, and within bone marrow). In B-cell precursor acute lymphoblastic leukemia (BCP-ALL), expression of CXCR4 was associated with higher capacity of leukemic blasts to seed into bone marrow niches. Aim of the study The study aimed at thorough analysis of CXCR4 expression on BCP-ALL blasts and correlation of CXCR4 expression with the expression of other antigens such as CD66c, CD34, CD10, CD38, CD20 and CD45 as well as with the levels of minimal residual disease on day 15. Patients and Methods The study group consisted of 198 consecutive children aged 0-18 years (median 4.4 years) treated for BCP-ALL in the centers of the Polish Pediatric Leukemia/Lymphoma Study Group. Bone marrow samples obtained at initial diagnosis were stained with monoclonal antibodies (CD58, CD66c, CD34, CD19, CD10, CD38, CD20, CD45, CXCR4) in two 8-color tubes and analyzed with multiparameter flow cytometry (BD FACSCanto II, Becton Dickinson, San Jose, CA, USA) according to the EuroFlow standard protocols. The expression of particular antigens on BCP-ALL blasts was defined by median fluorescence. In 177 patients the samples from day 15 were available and analyzed for the presence of minimal residual disease (MRD) with multicolor flow cytometry. Infinicyt software (Cytognos, Salamanca, Spain) was used for more detailed analyses of the flow cytometric data. Results The expression of CXCR4 in BCP-ALL was highly variable with median fluorescence ranging from 252 to 24 388 (median 4011). There was no obvious correlation of CXCR4 expression with immunophenotype and with  the expression of other analyzed markers (CD66, CD34, CD10, CD38 i CD45). The only borderline significant correlation found was between CXCR4 and CD20 expression. On day 15, 70 children (39%) demonstrated MRD levels below 0.1%, which is consistent with MRD-based low-risk group. Among these patients, 41 children had undetectable MRD already at this time point. In contrary, MRD levels > 10% were recorded in 21 patients (12%), who were stratified to high-risk group, accordingly. Maximal MRD levels recorded at day 15 were 85.6%. In remaining 86 children (49%), MRD levels at day 15 were in-between 0.1% and 10%, which reflects intermediate response to the treatment. There was no correlation between CXCR4 expression and MRD levels at day 15. Conclusion CXCR4 expression on BCP-ALL blasts is highly heterogeneous and is not associated with particular leukemia immunophenotype. Further analyses should characterize clinical features of leukemia and treatment response with regard to CXCR4 expression. The study was supported by Polish National Center of Science grant N N407 687040. Disclosures: No relevant conflicts of interest to declare.

Clonal Evolution Underlying the Progression from Myelodysplastic Syndromes to Ph-Positive Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5514-5514
Author(s):  
Masataka Taguchi ◽  
Tomoko Kohno ◽  
Hiroyuki Mishima ◽  
Hiroaki Taniguchi ◽  
Takeharu Kato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Myelodysplastic Syndromes ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Intron 1

Abstract Introduction: Myelodysplastic syndromes (MDS) are considered as a "stem cell disorders", in which hematopoietic stem cells and lineage-committed progenitor cells acquire genetic and epigenetic alterations and provide aberrant, clonal hematopoiesis, sometimes resulted in the progression to acute myeloid leukemia (Elias HK et al, Oncogene 2014). We previously reported a rare case of which the patient developed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) 2.5 years after being diagnosed with MDS (Kohno T et al, Br J Haematol 1996). p190 BCR-ABL1 mRNA was detected in the Ph+ALL cells. Metaphase cytogenetics showed the karyotypes: 46, XY, 20q- in MDS phase and 46, XY, t(9;22)(q34;q11), 20q- in ALL phase, indicating that MDS and Ph+ALL in this patient were of the same clonal origin. To uncover the detail of the clonal evolution, we analyzed bone marrow samples of MDS and Ph+ALL in this patient by targeted massively parallel sequencing with a panel of 154 genes including known driver genes of hematologic malignancies. Methods: Genomic DNAs (gDNAs) were extracted from the bone marrow mononuclear cells of MDS and Ph+ALL in this patient. Targeted sequencing was performed on the Illumina HiSeq2500 platform. Single nucleotide variants (SNVs) and small insertions and deletions (INDELs) were called using HaplotypeCaller of Genome Analysis Toolkit (GATK) version 3.4-46. We also attempted to detect the breakpoint of BCR-ABL1 translocation from the targeted sequencing data using the computational method, BreaKmer (Abo RP et al, Nucleic Acids Research 2015). The candidates of the mutations and structural variations were validated by amplicon-based deep sequencing and Sanger sequencing. Copy number variations were analyzed using Affymetrix CytoScan HD Array. Results: The mutations in ASXL1 and U2AF1 genes were identified in the MDS sample with variant allele frequencies (VAFs) of about 45%. At the progression of Ph+ALL, the mutations in SETBP1, SMC1A, and SLC5A8 genes were newly acquired while the ASXL1 and U2AF1 mutations were also identified with the same level of VAFs (about 50%) as the other mutations. VAFs of all of the mutations were decreased to about 20% after the chemotherapy for Ph+ALL, and then increased to about 40% at the recurrence of the disease. Furthermore, we identified the breakpoint of BCR-ABL1 translocation at intron 1 of ABL1 genes and intron 1 of BCR genes, that is the well-known cluster region, m-bcr, only among the samples of Ph+ALL. Copy number analysis confirmed that both MDS and Ph+ALL samples harbored the deletion of chromosome 20q. And the deletion of IKZF1 gene, which is frequently identified in Ph+ALL cases (Mullighan CG et al, Nature 2008), was not identified during the progression from MDS to Ph+ALL. These results demonstrated that the MDS clone harboring 20q- and ASXL1 and U2AF1 mutations acquired the mutations in SETBP1, SMC1A, and SLC5A8 genes and the p190 BCR-ABL1, resulted in the development of Ph+ALL in this patient. Conclusion: The alterations of SETBP1, SMC1A, and SLC5A8 genes are usually reported in myeloid malignancies (Makishima H et al, Nat Genet 2013, Kon A et al, Nat Genet 2013, Whitman SP et al, Blood 2008). Previous study in transgenic mouse demonstrated the distinct role of p190 BCR-ABL1 in the development of an ALL (Voncken JW et al, Blood 1995). Recapitulating this scenario, p190 BCR-ABL1 may play a critical role in the development of Ph+ALL from the MDS stem cells in this patient. This study may provide a new insight into the stem cell origin of MDS and the role of p190 BCR-ABL1 in the development of Ph+ALL. Disclosures No relevant conflicts of interest to declare.

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