| Abstract|| |
Rearrangements of the mixed lineage leukemia (MLL) gene at 11q23 commonly occur in infants with CALLA negative B lymphoblastic leukemia (B-ALL). Most often, these are detected by conventional karyotyping; however, fluorescent in-situ hybridization (FISH) with the help of a dual-color break-apart probe is used to identify cryptic translocations. When there is an MLL gene translocation, the usual FISH signal pattern is 1 red-1 green-1 yellow fusion signal pattern. We present a case of an infant with CALLA negative precursor B-ALL with a characteristic translocation t(4;11) (q21;q23), however, with an unusual MLL FISH signal pattern.
Keywords: B lymphoblastic leukemia, CALLA negative, conventional karyotyping, fluorescent in-situ hybridization, mixed lineage leukemia gene
|How to cite this article:|
Devi SG, Goyal M, Ramakrishna N, Murthy S S. CALLA negative precursor B lymphoblastic leukemia with MLL gene translocation and an unusual FISH signal pattern. Indian J Pathol Microbiol 2011;54:176-9
|How to cite this URL:|
Devi SG, Goyal M, Ramakrishna N, Murthy S S. CALLA negative precursor B lymphoblastic leukemia with MLL gene translocation and an unusual FISH signal pattern. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Nov 17];54:176-9. Available from: http://www.ijpmonline.org/text.asp?2011/54/1/176/77396
| Introduction|| |
Majority of the B lymphoblastic leukemias (B-ALLs) have cytogenetic abnormalities, some defining specific entities with unique phenotypic and prognostic features.  Structural rearrangements of the mixed lineage leukemia (MLL) gene at 11q23 are well documented recurring abnormalities in B-ALL/ Lymphoblastic lymphoma , occurring commonly in infants and less frequently in children and adults . [ 1], Conventional karyotyping detects most MLL related translocations with identification of its different partner chromosomes. , Fluorescent in-situ hybridization (FISH) using a dual-color break-apart probe is a useful technique to detect MLL related translocations in the interphase cells in a rapid and sensitive manner, when these are either cryptic or when the partner chromosomes are not the usual ones. , This probe is made of differentially labeled (red and green) DNA segments located on either side of the MLL breakpoint cluster region In normal cells, the two probes co-localize to produce two yellow fusion signals.  When there is a translocation involving the MLL gene, one fusion signal splits, resulting in a characteristic 1 red-1 green-1 yellow fusion signal pattern. 
We hereby present a case of CALLA negative precursor B-ALL with a characteristic translocation t(4;11) (q21;q23), however, with an unusual MLL FISH signal pattern.
| Case Report|| |
An 11-month-old male infant had complaints of fever, cough, and breathlessness for about 2 months. He had cervical lymphadenopathy and splenomegaly. Complete blood picture revealed hemoglobin of 7 g/dl, total leukocyte count of 1.9 × 10 5 /μ l and platelet count of 39 × 10 3 /μ l. Peripheral smear and bone marrow aspirate smears showed marked prominence of blasts, which were morphologically lymphoblastic and myeloperoxidase negative.
Immunophenotyping was done on peripheral blood using lyse/wash technique and was acquired on dual-laser four-color FACS Calibur (Becton Dickinson, San Jose, CA, USA). The results were analyzed using the CellQuest software utilizing both forward scatter/side scatter and CD45/side scatter gating strategies. The gated population of cells had a low forward and side scatter. CD45 expression was moderate. These cells expressed moderate CD19, moderate HLA-DR, dim surface CD22, dim nuclear terminal-deoxynucleotidyl transferase (TdT), and heterogeneous CD34. These cells were negative for CD10, CD38, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD33, and CD117 [Figure 1].
|Figure 1: The gated population of cells (red) show moderate CD45 positivity with expression of moderate CD19, HLA-DR, dim surface CD22, dim nuclear TdT, and heterogeneous CD34. These cells were negative for CD10, CD38, CD3, CD4, CD5, CD7, CD13, and CD33|
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FISH analysis was performed on interphase cells of peripheral blood using the locus specific identifier (LSI) MLL dual-color, break-apart rearrangement probe (Vysis, Abbott Park, IL, USA). The LSI MLL probe consists of a 350-kb portion centromeric of the MLL gene breakpoint cluster region (bcr) labeled in Spectrum Green and approximately 190 kb portion largely telomeric of the bcr labeled in Spectrum Orange. The denaturation and hybridization procedure was performed according to the manufacturer's instructions and counterstained with 4,6-diamino-2-phenyl-indole (DAPI) stain. Fluorescent signals were visualized under the Olympus BX41 microscope equipped with the CCD camera and CytoVision Software (Applied Imaging, Inc., San Jose, CA, USA ) and 200 interphase nuclei were analyzed. Nuclei with ambiguous signals were excluded from analysis. An atypical pattern of 0 red-1 green-1 yellow fusion signal pattern was observed in 88% of the cells, suggesting that the patient had MLL gene rearrangement with possible 3´ MLL gene deletion [Figure 2].
|Figure 2: Representative interphase FISH using MLL dual-color break-apart probe shows MLL gene rearrangement with 0 red-1 green-1 yellow fusion signal pattern. Yellow or red-green fusion signal indicates an intact MLL gene, whereas green signal indicates the separated MLL gene with 3' MLL gene deletion (loss of red signal)|
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Simultaneously, peripheral blood was cultured in Marrowmax medium without phytohaemagglutinin (Invitrogen, Carlsbad, CA, USA), followed by GTG banding. An analysis of 20 metaphases showed a balanced reciprocal translocation between long arms of chromosomes 4 and 11, between the region q21 and q23, respectively, with the karyotype 46,XY,t(4;11)(q21;q23) [Figure 3]. However, there was no deletion identified.
|Figure 3: Karyotype showing balanced translocation of 46,XY,t(4;11)(q21;q23)|
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A final diagnosis of CALLA negative precursor B-ALL with t(4;11)(q21;q23); MLL rearranged was rendered. The patient was lost to follow-up before he could complete the induction phase of chemotherapy.
| Discussion|| |
The MLL gene on chromosome11q23 has an important role in normal axial-skeletal and hematopoietic developments.  MLL is involved in a wide spectrum of acute leukemias and is implicated in more than 80 different 11q23 translocations.  Most common translocations are t(4;11)(q21;q23), t(11;19)(q23;p13), and t(9;11)(p22;q23), and are associated with B-ALL, T-ALL, and acute myeloid leukaemia, respectively. , B-ALL with MLL gene rearrangements are known to occur commonly in infants under 1 year of age, with marked leukocytosis (>1 × 10 5 /μl), CNS involvement, and organomegaly. ,, Morphologically, these are characteristically lymphoblastic; however, they rarely present as distinct lymphoblastic and monoblastic populations, when these are considered as B/myeloid leukemias.  Immunophenotypically, these are typically CD19+, CD10 negative and are considered pro-B type. , These cells characteristically are CD24 negative, CD15+, CD65w+ and also positive for chondroitin sulfate proteoglycan neural glial antigen 2 (NG2). ,
Interphase FISH using a dual-color break-apart probe to identify MLL related translocations showed a 0 red-1 green-1 yellow fusion signal pattern, consistent with the presence of MLL related translocation due to the splitting of fusion signal. The loss of red signal implies deletion of 3´ region of MLL. Few cases of MLL gene rearrangements with atypical FISH signal patterns, such as loss of either green or red signal, suggesting microdeletions of the 5´ and 3´ MLL regions, respectively, have been reported. ,,,,
Our case revealed a balanced translocation of t(4;11)(q21;q23), representing the fusion of MLL/AF4 genes, without any apparent deletion. However, the fact that the 3´ MLL region is deleted in our case could have been definitely established either by FISH using the specific dual-color dual-fusion probe or by performing metaphase-FISH.  There is evidence that the deletion of 3´ region of MLL gene correlated with deletion of at least 190 kb of the region that is telomeric to the gene and this loss of genomic material is below the level of the resolution of classical cytogenetic methods.  Some reports have suggested that the loss of the 3´ red signal arises from a concurrent deletion event, in which the retention of 5´ MLL is consistent with the preservation of the more important of the two fusion products. ,, They also demonstrated the presence of repetitive DNA Alu sequences in the vicinity of breakpoint regions in MLL genes and their flanking regions, and suggested that these sequences may facilitate the generation of submicroscopic deletions.  Such deletions could lead to the loss of one or more genes, and the associated haploinsufficiency may result in the differences in clinical behavior. 
Analysis of MLL gene rearrangements in pediatric ALL represents an indispensable prerequisite for the accurate classification of the leukemic disease because children with MLL-rearranged ALL usually have an increased risk of treatment failure and a poor prognosis. ,, There is a lot uncertainty as to the prognostic implications of MLL submicroscopic deletions in terms of low survival, less duration of complete remission rates, and a prognosis worse than that for the usual MLL translocations. More cases have to be studied with their follow-up to resolve these issues.
| Acknowledgments|| |
We would like to thank Dr. Salil N. Vaniawala, PhD, from Gene Lab, Surat, for performing the karyotype, providing the technical details and image. We also extend our thanks to Ms. M. Padma and Mr. N. Koteswara Rao for performing FISH and immunophenotyping, respectively.
| References|| |
|1.||Borowitz MJ, Chan JK. B-lymphoblastic leukemia/ lymphoma with recurrent genetic abnomalities. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. editors. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4 th ed. Lyon, PA: IARC; 2008. p. 171-5. |
|2.||Attarbaschi A, Mann G, Konig M, Steiner M, Strehl S, Schreiberhuber A, et al. Mixed lineage leukemia rearranged childhood Pro-B and CD10-Negative Pre-B acute lymphoblastic leukemia constitute a distinct clinical entity. Clin Cancer Res 2006;12:2988-94. |
|3.||Wolff DJ, Bagg A, Cooley LD, Dewald GW, Hirsch BA, Jacky PB, et al. Association for Molecular Pathology Clinical Practice Committee; American College of Medical Genetics Laboratory Quality Assurance Committe. Guidance for fluorescence in situ hybridization testing in hematologic disorders. J Mol Diagn 2007;9:134-43. |
|4.||Nordgren A, Heyman M, Sahlen S, Schoumans J, Soderhall S, Nordenskjold M, et al. Spectral karyotyping and interphase FISH reveal abnormalities not detected by conventional G-banding. Eur J Haematol 2002;68:31-41. |
|5.||Li ZY, Liu DP, Liang CC. New insight into the molecular mechanisms of MLL-associated leukemia. Leukemia 2005;19:183-90. |
|6.||Pais A, Kadam PA, Raje G, Sawant M, Kabre S, Jain H, et al. Identification of various MLL gene aberrations that lead to MLL gene mutation in patients with acute lymphoblastic leukemia (ALL) and infants with acute leukemia. Leukemia Res 2005;29:517-26. |
|7.||Gulten T, Yakut T, Gunes AM, Demirkaya M, 5¢ MLL Gene Deletion in a Case with Childhood Acute Lymphoblastic Leukemia. Lab Medicine 2010;41:83-6. |
|8.||Barber KE, Ford AM, Harris RL, Harrison CJ, Moorman AV. MLL translocations with concurrent 3' deletions: Interpretation of FISH results. Genes Chromosomes Cancer 2004;41:266-71. |
|9.||Kolomietz E, Al Maghrabi J, Brennan S, Karaskova J, Minkin S, Lipton J, et al. Primary chromosomal rearrangements of leukemia are frequently accompanied by extensive submicroscopic deletions and may lead to altered prognosis. Blood 2001;97:3581-8. |
|10.||König M, Reichel M, Marschalek R, Haas OA, Strehl S. A highly specific and sensitive fluorescence in situ hybridization assay for the detection of t(4;11)(q21;q23) and concurrent submicroscopic deletions in acute leukemias. Br J Haematol 2002;116:758-64. |
Sandhya G Devi
Department of Laboratory Medicine, Indo-American Cancer Institute and Research Centre, Road No.14, Banjara Hills, Hyderabad - 500 034
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]