Abstract
Chromosome abnormalities that usually define high-risk acute lymphoblastic leukemia are the t(9;22)/ breakpoint cluster region protein-Abelson murine leukemia viral oncogene homolog 1, hypodiploid with < 44 chromosomes and 11q23/ myeloid/lymphoid leukemia gene rearrangements. The spectrum of acute lymphoblastic leukemia genetic abnormalities is nevertheless rapidly expanding. Therefore, newly described chromosomal aberrations are likely to have an impact on clinical care in the near future. Recently, the rare intrachromosomal amplification of chromosome 21 started to be considered a high-risk chromosomal abnormality. It occurs in approximately 2-5% of pediatric patients with B-cell precursor acute lymphoblastic leukemia. This abnormality is associated with a poor outcome. Hence, an accurate detection of this abnormality is expected to become very important in the choice of appropriate therapy. In this work the clinical and molecular cytogenetic evaluation by fluorescence in situ hybridization of a child with B-cell precursor acute lymphoblastic leukemia presenting the rare intrachromosomal amplification of chromosome 21 is described.
Leukemia, lymphoid; In situ hybridization, fluorescence; Gene amplification; Leukemia, B-cell; Chromosomes, human, pair 21/genetics; Transcription factors; Case reports
Introduction
Cytogenetic techniques have galvanized efforts to understand the biology of chromosomal
changes in childhood acute lymphoblastic leukemia (ALL) and to identify specific
alterations that can predict treatment outcome(11. Pui CH, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet.
2008;371(9617):1030-43.). Several clinically relevant abnormalities are used in
risk stratification for treatment within the context of clinical trials(22. Rand V, Parker H, Russell LJ, Schwab C, Ensor H, Irving J, et al.
Genomic characterization implicates iAMP21 as a likely primary genetic event in
childhood B-cell precursor acute lymphoblastic leukemia. Blood.
2011;117(25):6848-55.). Some chromosome abnormalities that
have defined ALL as very high risk are those with t(9;22)/breakpoint cluster region
protein-Abelson murine leukemia viral oncogene homolog 1, those that are hypodiploid
with < 44 chromosomes, and with 11q23/myeloid/lymphoid leukemia (MLL) gene
rearrangements(33. Heerema NA, Raetz EA, Carroll AJ, Borowitz MJ, Devidas VM,
Gastier-Foster JM, Larsen EC, Loh ML, Mattano LA, Maloney KW, Willman CL, Wood BL,
Winick N, Hunger SP, Carroll WL. 739 iAMP21 is associated with inferior outcomes in
children with Acute Lymphoblastic Leukemia (ALL) on contemporary Children's Oncology
Group (COG) Studies Program. Blood (ASH Annual Meeting Abstracts) [Internet].
2011[cited 2012 Mar 21];118(21): abstract 739. Available from:
http://abstracts.hematologylibrary.org/cgi/content/abstract/118/21/739?maxtoshow=&hits=10&RESULTFORMAT=&fulltext=heerema+na&searchid=1&FIRSTINDEX=0&volume=118&issue=21&resourcetype=HWCIT
http://abstracts.hematologylibrary.org/c...
). The
spectrum of genetic abnormalities described in ALL is rapidly expanding; therefore,
newly described aberrations are likely to have an impact on clinical care in the near
future(44. Moorman AV. The clinical relevance of chromosomal and genomic
abnormalities in B-cell precursor acute lymphoblastic leukaemia. Blood Rev.
2012;26(3):123-35.).
Intrachromosomal amplification of chromosome 21 (iAMP21) is a rare high-risk chromosomal abnormality that can occur in approximately 2-5% of pediatric patients with B-cell precursor ALL (BCP-ALL)(44. Moorman AV. The clinical relevance of chromosomal and genomic abnormalities in B-cell precursor acute lymphoblastic leukaemia. Blood Rev. 2012;26(3):123-35.,55. Ma SK, Wan TS, Cheuk AT, Fung LF, Chan GC, Chan SY, et al. Characterization of additional genetic events in childhood acute lymphoblastic leukemia with TEL/AML1 gene fusion: a molecular cytogentics study. Leukemia. 2001;15(9):1442-7.). This abnormality has been associated with a poor outcome in patients treated by a standard protocol(22. Rand V, Parker H, Russell LJ, Schwab C, Ensor H, Irving J, et al. Genomic characterization implicates iAMP21 as a likely primary genetic event in childhood B-cell precursor acute lymphoblastic leukemia. Blood. 2011;117(25):6848-55.,66. Moorman AV, Richards SM, Robinson HM, Strefford JC, Gibson BE, Kinsey SE, Eden TO, Vora AJ, Mitchell CD, Harrison CJ; UK Medical Research Council (MRC)/National Cancer Research Institute (NCRI) Childhood Leukaemia Working Party (CLWP). Prognosis of children with acute lymphoblastic leukemia (ALL) and intrachromosomal amplification of chromosome 21 (iAMP21). Blood. 2007;109(6):2327-30.). Therefore, an accurate detection of this abnormality is very important to the choice of appropriate therapy(77. Harrison CJ. Acute lymphoblastic leukemia. Clin Lab Med. 2011;31(4):631-47.).
Here, a clinical and molecular cytogenetic analysis of a child with BCP-ALL and presenting the rare iAMP21 abnormality is described.
Case report
An 11-year-old girl with a two-month history of joint pain in both hands and shoulders that evolved to arthritis was admitted to the Onco-Hematology Pediatric Service of Hospital Estadual Bauru, São Paulo, Brazil. A physical examination revealed that the patient did not present enlarged palpable lymph nodes in the cervical, submandibular, axillary or inguinal regions. No signs of musculoskeletal abnormalities, including inflammatory joint arthritic changes, were noted. Examinations of the cardiovascular and respiratory systems were also normal. The hemoglobin level was low (11 g/dL), white blood cell count was also low (3 x 109/L) with no blast cells, platelet count was 165 x 109/L and lactate dehydrogenase (LDH) was 23 U/L. The morphologic evaluation of the bone marrow showed 95% blast cells with lymphoid characteristics compatible with French-American-British (FAB) classification L1. Flow cytometry revealed a population with 71% of blast cells that expressed CD79a, CD19, CD20, CD38, CD58, CD66c heterogeneously, low expressions for CD10, CD13, CD22, and negative expressions for cCD3, sCD3, cCD7, CD45, CD34, CD15, CD33, cIgM, and myeloperoxidase (MPO) thus compatible with BCP-ALL. The patient was initially treated according to the ALL-IC-BFM2009 protocol for medium risk(88. Attarbaschi A, Mann G, Panzer-Grümayer R, Röttgers S, Steiner M, König M, et al. Minimal residual disease values discriminate between low and high relapse risk in children with B-cell precursor acute lymphoblastic leukemia and an intrachromosomal amplification of chromosome 21: the Austrian and German acute lymphoblastic leukemia Berlin-Frankfurt-Munster (ALL-BFM) trials. J Clin Oncol. 2008;26(18):3046-50.). Peripheral blood showed good treatment response to prednisone on Day 8, and bone marrow examination at the end of the induction therapy showed complete remission. The patient is clinically well without any evidence of disease five months after diagnosis.
The patient's bone marrow sample was sent for cytogenetic analysis, at the time of diagnosis, to the Cytogenetics Laboratory of the Bone Marrow Unit, National Cancer Institute (INCA). The 24-hour culture technique was unsuccessful in obtaining metaphase cells for chromosome analysis(99. Testa JR, Misawa S, Oguma N, Van Sloten K, Wiernik PH. Chromosomal alterations in acute leukemia patients studied with improved culture methods. Cancer Res. 1985;45(1):430-4.). Fluorescence in situ hybridization (FISH) was performed on interphase cells using the commercially available LSI ETV6/RUNX1 ES dual color translocation probe, LSI MLL dual color break apart rearrangement probe, and the LSI BCR/ABL ES dual color translocation probe (All from Abbott Diagnostics, Maidenhead, United Kingdom), according to the manufacturer's instructions. Two-hundred interphase cells were scored for each probe.
Interphase FISH (iFISH) analysis revealed no ETV6/RUNX1 fusion signals and 6 to 10 signals for the RUNX1 probe in 75% of the analyzed nuclei in this child (Figure 1A & 1B). FISH analyses with MLL and BCR/ABL probes showed normal results.
iFISH with LSI TEL/AML1 dual fusion probe; A) iFISH detected 8 in tandem copies of RUNX1 gene in the present case; B) Negative control for the TEL/AML1 probe showing a normal cell
Discussion
Patients with iAMP21 are characterized by a low white blood cell count, older age (> 5 years old, with a median age of 9-10 years), FAB L1 classification, and common/pre-B cell immunophenotype(44. Moorman AV. The clinical relevance of chromosomal and genomic abnormalities in B-cell precursor acute lymphoblastic leukaemia. Blood Rev. 2012;26(3):123-35.,66. Moorman AV, Richards SM, Robinson HM, Strefford JC, Gibson BE, Kinsey SE, Eden TO, Vora AJ, Mitchell CD, Harrison CJ; UK Medical Research Council (MRC)/National Cancer Research Institute (NCRI) Childhood Leukaemia Working Party (CLWP). Prognosis of children with acute lymphoblastic leukemia (ALL) and intrachromosomal amplification of chromosome 21 (iAMP21). Blood. 2007;109(6):2327-30.,77. Harrison CJ. Acute lymphoblastic leukemia. Clin Lab Med. 2011;31(4):631-47.). In a multivariate analysis, Moorman et al.(66. Moorman AV, Richards SM, Robinson HM, Strefford JC, Gibson BE, Kinsey SE, Eden TO, Vora AJ, Mitchell CD, Harrison CJ; UK Medical Research Council (MRC)/National Cancer Research Institute (NCRI) Childhood Leukaemia Working Party (CLWP). Prognosis of children with acute lymphoblastic leukemia (ALL) and intrachromosomal amplification of chromosome 21 (iAMP21). Blood. 2007;109(6):2327-30.) observed that the presence of the iAMP21 was an independent indicator of poor event-free and overall survival. Studies have identified iAMP21 as one of the most significant, recently discovered abnormalities in ALL in which genotype has a direct impact on treatment(44. Moorman AV. The clinical relevance of chromosomal and genomic abnormalities in B-cell precursor acute lymphoblastic leukaemia. Blood Rev. 2012;26(3):123-35.,66. Moorman AV, Richards SM, Robinson HM, Strefford JC, Gibson BE, Kinsey SE, Eden TO, Vora AJ, Mitchell CD, Harrison CJ; UK Medical Research Council (MRC)/National Cancer Research Institute (NCRI) Childhood Leukaemia Working Party (CLWP). Prognosis of children with acute lymphoblastic leukemia (ALL) and intrachromosomal amplification of chromosome 21 (iAMP21). Blood. 2007;109(6):2327-30.,77. Harrison CJ. Acute lymphoblastic leukemia. Clin Lab Med. 2011;31(4):631-47.).
iAMP21 was first detected by FISH using the ETV6/RUNX1 probe(77. Harrison CJ. Acute lymphoblastic leukemia. Clin Lab Med. 2011;31(4):631-47.). iAMP21 can be observed by conventional cytogenetics as a gross abnormality of chromosome 21. This finding is usually observed by FISH analysis, in metaphase, as one signal located on a normal chromosome 21, while the others are seen in tandem duplication along an abnormal chromosome 21(22. Rand V, Parker H, Russell LJ, Schwab C, Ensor H, Irving J, et al. Genomic characterization implicates iAMP21 as a likely primary genetic event in childhood B-cell precursor acute lymphoblastic leukemia. Blood. 2011;117(25):6848-55.). When ETV6/ RUNX1 probes are used, patients carrying the iAMP21 usually show two normal copies of the ETV6 signal and multiple RUNX1 signals (three or more additional signals), and are negative for the ETV6-RUNX1 fusion gene. This abnormal chromosome 21 may vary according to its morphological form. In interphase, the signals are clustered together, except for one signal representing the normal chromosome 21 that is usually located apart from the others(22. Rand V, Parker H, Russell LJ, Schwab C, Ensor H, Irving J, et al. Genomic characterization implicates iAMP21 as a likely primary genetic event in childhood B-cell precursor acute lymphoblastic leukemia. Blood. 2011;117(25):6848-55.,77. Harrison CJ. Acute lymphoblastic leukemia. Clin Lab Med. 2011;31(4):631-47.).
Recent studies have shown that iAMP21 is mostly found as a single abnormality, suggesting that it is likely to be a primary genetic event given the lack of any other consistent abnormality outside of chromosome 21(22. Rand V, Parker H, Russell LJ, Schwab C, Ensor H, Irving J, et al. Genomic characterization implicates iAMP21 as a likely primary genetic event in childhood B-cell precursor acute lymphoblastic leukemia. Blood. 2011;117(25):6848-55.). Although RUNX1 is a well-known leukemia-related gene, it does not appear to be the target of iAMP21. iAMP21 is thought to arise through a breakage-fusion-bridge cycle mechanism(1010. Reichard KK, Kang H, Robinett S. Pediatric B-lymphoblastic leukemia with RUNX1 amplification: clinicopathologic study of eight cases. Mod Pathol. 2011;24(12):1606-11.). These breaks were recently determined by aCGH and long distance inverted (LDI) polymerase chain reaction (PCR) that pointed to an initiating event involving chromothripsis of chromosome 21. This would explain the unexpected nature of the PDE9A rearrangements, the cytogenetic heterogeneity of the iAMP21 marker chromosomes, and the complexity of some array profiles. The common region of amplification on chromosome 21 was confined to a 5.1-mb region that included the RUNX1, miR-802, genes mapping to the Down syndrome critical region, and also telomeric breakpoints(1111. Sinclair PB, Parker H, An Q, Rand V, Ensor H, Harrison CJ, et al. Analysis of a breakpoint cluster reveals insight into the mechanism of intrachromosomal amplification in a lymphoid malignancy. Hum Mol Genet. 2011;20(13):2591-602.).
Cytogenetic analysis sometimes is hampered by poor mitotic index and poor quality of chromosome preparations, problems that are frequent in ALL studies, as well as the presence of cryptic abnormalities that mask the detection of specific chromosome aberrations by conventional techniques(44. Moorman AV. The clinical relevance of chromosomal and genomic abnormalities in B-cell precursor acute lymphoblastic leukaemia. Blood Rev. 2012;26(3):123-35.,55. Ma SK, Wan TS, Cheuk AT, Fung LF, Chan GC, Chan SY, et al. Characterization of additional genetic events in childhood acute lymphoblastic leukemia with TEL/AML1 gene fusion: a molecular cytogentics study. Leukemia. 2001;15(9):1442-7.). The FISH technique is routinely used to complement cytogenetic studies in pediatric ALL. Our observation underscores the value of FISH analysis as an efficient and fast tool to detect a variety of biomarkers in a single experiment, providing important insights for the management of children with leukemia.
In this work, FISH analysis using the ETV6/RUNX1 probe allowed the identification of a rare chromosome abnormality with important prognostic impact, the iAMP21. Although iAMP21 has been associated with poor prognosis, new therapies with more intensive treatment may leave behind the prognostic importance of the genetic abnormality.
The authors acknowledge with gratitude Mariana Tavares de Souza, Dr. Tarsis Paiva Vieira, Roberto Rodrigues Capela de Matos, and Dr. Eliana Abdelhay.
This work was supported by CAPES (project no. 301/08); Pro-Vita is a not-for-profit organization; CNPq (project no. 473878/2011-9) and FAPERJ (project no. E-26/110:899/2009); the International Outreach Program and the American Lebanese Syrian Associated Charities (ALSAC), St. Jude Children's Research Hospital (Memphis, TN); and a Center of Excellence Grant, State of Tennessee.
References
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1Pui CH, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet. 2008;371(9617):1030-43.
-
2Rand V, Parker H, Russell LJ, Schwab C, Ensor H, Irving J, et al. Genomic characterization implicates iAMP21 as a likely primary genetic event in childhood B-cell precursor acute lymphoblastic leukemia. Blood. 2011;117(25):6848-55.
-
3Heerema NA, Raetz EA, Carroll AJ, Borowitz MJ, Devidas VM, Gastier-Foster JM, Larsen EC, Loh ML, Mattano LA, Maloney KW, Willman CL, Wood BL, Winick N, Hunger SP, Carroll WL. 739 iAMP21 is associated with inferior outcomes in children with Acute Lymphoblastic Leukemia (ALL) on contemporary Children's Oncology Group (COG) Studies Program. Blood (ASH Annual Meeting Abstracts) [Internet]. 2011[cited 2012 Mar 21];118(21): abstract 739. Available from: http://abstracts.hematologylibrary.org/cgi/content/abstract/118/21/739?maxtoshow=&hits=10&RESULTFORMAT=&fulltext=heerema+na&searchid=1&FIRSTINDEX=0&volume=118&issue=21&resourcetype=HWCIT
» http://abstracts.hematologylibrary.org/cgi/content/abstract/118/21/739?maxtoshow=&hits=10&RESULTFORMAT=&fulltext=heerema+na&searchid=1&FIRSTINDEX=0&volume=118&issue=21&resourcetype=HWCIT -
4Moorman AV. The clinical relevance of chromosomal and genomic abnormalities in B-cell precursor acute lymphoblastic leukaemia. Blood Rev. 2012;26(3):123-35.
-
5Ma SK, Wan TS, Cheuk AT, Fung LF, Chan GC, Chan SY, et al. Characterization of additional genetic events in childhood acute lymphoblastic leukemia with TEL/AML1 gene fusion: a molecular cytogentics study. Leukemia. 2001;15(9):1442-7.
-
6Moorman AV, Richards SM, Robinson HM, Strefford JC, Gibson BE, Kinsey SE, Eden TO, Vora AJ, Mitchell CD, Harrison CJ; UK Medical Research Council (MRC)/National Cancer Research Institute (NCRI) Childhood Leukaemia Working Party (CLWP). Prognosis of children with acute lymphoblastic leukemia (ALL) and intrachromosomal amplification of chromosome 21 (iAMP21). Blood. 2007;109(6):2327-30.
-
7Harrison CJ. Acute lymphoblastic leukemia. Clin Lab Med. 2011;31(4):631-47.
-
8Attarbaschi A, Mann G, Panzer-Grümayer R, Röttgers S, Steiner M, König M, et al. Minimal residual disease values discriminate between low and high relapse risk in children with B-cell precursor acute lymphoblastic leukemia and an intrachromosomal amplification of chromosome 21: the Austrian and German acute lymphoblastic leukemia Berlin-Frankfurt-Munster (ALL-BFM) trials. J Clin Oncol. 2008;26(18):3046-50.
-
9Testa JR, Misawa S, Oguma N, Van Sloten K, Wiernik PH. Chromosomal alterations in acute leukemia patients studied with improved culture methods. Cancer Res. 1985;45(1):430-4.
-
10Reichard KK, Kang H, Robinett S. Pediatric B-lymphoblastic leukemia with RUNX1 amplification: clinicopathologic study of eight cases. Mod Pathol. 2011;24(12):1606-11.
-
11Sinclair PB, Parker H, An Q, Rand V, Ensor H, Harrison CJ, et al. Analysis of a breakpoint cluster reveals insight into the mechanism of intrachromosomal amplification in a lymphoid malignancy. Hum Mol Genet. 2011;20(13):2591-602.
Publication Dates
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Publication in this collection
2013
History
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Received
24 Apr 2013 -
Accepted
01 Aug 2013