Journal of Cancer Research and Therapeutics

: 2013  |  Volume : 9  |  Issue : 3  |  Page : 493--496

Molecular diagnosis of lymphoblastic leukemia

Kalal Iravathy Goud1, Seetha Dayakar1, S. V. S. S. Prasad2, Koteshwar N Rao3, Amina Shaik3, S Vanjakshi3,  
1 Department of Molecular Biology and Cytogenetics, Apollo Health City, Jubilee Hills, Hyderabad, India
2 Department of Medical Oncology, Apollo Health City, Jubilee Hills, Hyderabad, India
3 Department of Haematology, Apollo Health City, Jubilee Hills, Hyderabad, India

Correspondence Address:
Kalal Iravathy Goud
Department of Molecular Biology and Cytogenetics, Apollo Health City, Jubilee Hills, Hyderabad-500 033


The mixed lineage leukemia (MLL) gene at chromosome band 11q23 is commonly involved in reciprocal translocations that is detected in acute leukemia. The MLL gene, coomonly known as mixed lineage leukemia or myeloid lymphoid leukemia, has been independently identified and cloned from the 11q23 breakpoint of acute leukemia. We describe a patient with acute lymphoblastic leukemia whose cells had shown reciprocal translocation between short arm (p21) of chromosome 2 and long arm (q23) of chromosome number 11 [t(2;11) (p21;q23)] by cytogenetic analysis. Fluorescence in situ hybridization analysis (FISH) was also performed for reconfirmation with a probe for MLL which showed split signals, hybridizing to both the derivative 2 and 11 chromosomes. Our study confirmed FISH as the most suitable assay for detecting MLL rearrangements because of its sensitivity and speed. It recommended that FISH should be used as complementary to conventional cytogenetic analysis. In conclusion, evaluation of the t(2;11)(p21;q23) was done by molecular clarification and flow cytometry.

How to cite this article:
Goud KI, Dayakar S, Prasad S, Rao KN, Shaik A, Vanjakshi S. Molecular diagnosis of lymphoblastic leukemia.J Can Res Ther 2013;9:493-496

How to cite this URL:
Goud KI, Dayakar S, Prasad S, Rao KN, Shaik A, Vanjakshi S. Molecular diagnosis of lymphoblastic leukemia. J Can Res Ther [serial online] 2013 [cited 2020 Oct 20 ];9:493-496
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Full Text


Reciprocal chromosomal translocations are recurrent features of many hematological malignancies. The cloning of the genes located at the breakpoints of chromosomal translocations in leukemia and lymphoma has led to the identification of new genes involved in carcinogenesis. Molecular studies of the breakpoint of several translocations involving chromosomal band 11q23 led to the cloning of a gene that was named as mixed lineage leukemia or myeloid lymphoid leukemia (MLL). [1],[2] The MLL gene at chromosome band 11q23 is commonly involved in reciprocal translocations that are detected in acute leukemia. MLL is a ubiquitously expressed 3969 amino-acid (aa) nuclear protein whose function is incompletely understood. [1] This gene is also known as ALL1, HTRX, HRX, or TRX1. MLL abnormalities can be divided in two categories. The first category consists of MLL rearrangements, usually as translocations or insertions, some of them are cryptic, leading to fusion genes with a large number of partners. [3],[4] The main aim of this study is to emphasize the importance of modern molecular techniques and include them in the battery of investigations for diagnosis and treatment of acute leukemia.

 Case Report

A 1-year-old infant presented with fever, upper respiratory tract infection since 1 week. On examination, revealed gross pallor and mild splenomegaly, no lymphadenopathy. Complete blood picture revealed hemoglobin of 8 g/dL, leukocyte count of 1.1 × 10 3 /uL, platelet count of 15 × 10 3 /uL, and 40% blasts. After taking an informed consent approximately 3 ml of bone marrow aspirate was collected from iliac crest and 1 ml of the bone marrow was distributed in a sodium heparin vacutainer for cytogenetics analysis and remaining 2 ml was collected in an Ethylene diamine tetraacetic acid (EDTA) vial for bone marrow examination and immnuophenotyping studies. Cytogenetic analysis of the patient was performed at Molecular Biology and Cytogenetics lab, Apollo Hospitals, Hyderabad. A total of 24 and 48 h unstimulated cultures were set was using standard cytogenetic protocol, Moorehead et al. [5] A Geimsa trypsin (GTG)-banded chromosome study was performed using standard protocol. A total of 20 metaphases were scored under light transmission microscope and Leica CW4000 karyotyping soft ware was used for cytogenetic analysis. Metaphases were karyotyped according to International System for Human Cytogenetic Nomenclature criteria, Mitelman. [6]

For further confirmation, MLL gene rearrangement studies by fluorescence in-situ hybridization (FISH) analysis was also performed using the same 24 h unstimulated cultures. FISH analysis was performed using the commercially available critical region probes (Kreatech-Poseidon Diagnostics, Amsterdam, Netherlands, KBI probes). Microscopy was carried out using an Olympus Fluorescence Microscope-BX61 (Israel) equipped with single bandpass filters for Spectrum Green, Spectrum Orange, and DAPI.

Immunophenotyping was done on bone marrow aspirate sample using lyse/wash technique and was acquired data on single Laser Five color multi parametric Beckman coulter flow cytometer (Cytomics-FC500). The results were analyzed using the CXP software utilizing side scatter/forward scatter/CD45 gating strategies.

Bone marrow aspirate smears showed 77% blasts and blasts were negative for both myeloperoxidase and PAS stains and were diagnosed as acute lymphoblastic leukemia (ALL).

For cytogenetic analysis a total of 20 metaphases were examined from unstimulated cultures. The 15 metaphases showed reciprocal translocation between short arm (p21) of chromosome 2 and long arm (q23) of chromosome 11, 46, XY, t(2;11)(p21;q23)] and the remaining five metaphases showed normal male karyotype 46, XY [Figure 1]a and b. The FISH analysis on the unstimulated cultures showed one fusion and break apart signal (single red and single green separated) indicating the MLL gene rearrangement on chromosome number 11. Among the 500 cells examined 300 cells showed one fusion and break apart signal (single red and single green separated) indicating the MLL gene rearrangement and 200 cells showed intact fusion signals (yellow signals) indicating normal cells. The break part signals indicated the MLL gene rearrangements [Figure 2]a and b.{Figure 1}{Figure 2}

The results of immunophenotyping were analyzed using the CXP software utilizing side scatter/forward scatter/CD45 gating strategies. The gated population of cells had a low forward and side scatter with moderate CD45 expression. These expressed moderate CD19, dim cytoplasmic CD79a, dim surface CD22, HLADR, Tdt, CD34, and dim to moderate CD15 expression. These cells were negative for CD10, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD14, CD33, CD117, and cytoplasmic MPO. Immunophenotypically, the case has been diagnosed as CALLA/CD10 negative, B- ALL with CD15 expression [Figure 3]a and b.{Figure 3}


MLL gene rearrangements are signs of a bad prognosis in childhood ALL. Translocations are the most common rearrangements detected, while deletions are rarely seen. However, it was not easy to predict the prognostic effect of the 3′ deletion of the MLL gene without a translocation. Conventional cytogenetics and FISH analysis are the first choices and are complementary to one another, but FISH is a more sensitive method for detecting chromosomal breakpoints.

The majority of 11q23 translocations disrupt to a MLL gene, which is a poor prognostic factor for acute lymphoid and myeloid leukemia. However, the presence of 11q23 abnormality does not always correlate with MLL rearrangement. In previous studies, rearrangements of the MLL gene locus were reported in approximately 1%-2% of childhood and 60%-80% of infant ALL cases. [7] The most common rearrangements of the MLL gene are reciprocal chromosomal translocations, and their prognostic relevance is well-established. MLL is involved in a wide spectrum of acute leukemia's 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 leukemia, respectively. The translocation t(2;11)(p21;q23) is more frequently seen in myelodysplastic syndrome and acute myeloid leukemia but was also reported in acute lymphoid leukemia by Gozzetti et al., [8] which is similar to our findings.

The balanced translocation of t(2;11)(q21;q23), [Figure 3]a and b which was seen by cytogenetics analysis was further confirmed by FISH by using the break apart MLL probe. The translocation in FISH was represented by the spilt signal [Figure 1]b. Immunophenotypically, the case has been diagnosed as CALLA/CD10 negative, ALL with CD15 expression which is more frequently seen in patients with MLL gene rearrangements as seen in our present study. Schwartz et al., [9] also reported MLL gene rearrangement with patients disclosing a CD65s+ and/or CD15+ immunophenotype.

The MLL fusion genes usually occur in tumors of specific hematological lineages, leading to the hypothesis that the MLL partner plays a critical role in determining the disease phenotype (for example, MLL-AFX1 in T-ALL, MLL-AF4 in B lineage ALL, MLL-EEN in AML, MLL-ENL in ALL/AML. This suggests that the fusion protein affects the differentiation of the hematopoietic pluripotent stem cells or the lymphoid or myeloid committed stem cells.

Improvements in chemotherapy and supportive care as well as insights into the molecular mechanisms have contributed to the advances. In addition, bone marrow transplants provide another treatment option. Understanding the treatment strategy for the MLL gene rearrangement positive patients is very important aspect. Infants with ALL have a particularly high risk of treatment failure. Treatment failure is most common in infants younger than 6 months and in those with extremely high presenting leukocyte counts and/or a poor response to a prednisone prophase. [10] Infants with ALL can be divided into two subgroups on the basis of the presence or absence of translocations that involve the MLL gene located at chromosome 11q23. Approximately, 80% of infants with ALL have an MLL gene rearrangement. The rate of MLL gene translocations is extremely high in infants younger than 6 months; from 6 months to 1 year the incidence of MLL translocations decreases but remains higher than that observed in older children.

Infants with leukemia and MLL translocations have very high white blood cell (WBC) counts, increased incidence of central nervous system involvement, and a poor outcome. Blasts from infants with MLL translocations are typically CD10 negative and express high levels of FLT3. Conversely, infants whose leukemic cells show a germline MLL gene configuration frequently present with CD10-positive precursor-B immunophenotype. These infants have a significantly better outcome than infants with ALL characterized by MLL translocations. [10],[11] Infants diagnosed within the first month of life have higher WBC counts, higher incidence of MLL translocations, significantly higher relapse rate, and poorer overall survival compared with infants older than 1 month at diagnosis.

In the present case, the child completed the chemotherapy (Vincistine Injection. Daunomycin Injection. Zexate intrathecal Injection. Decadron along with Injection. Zofer), during the course of treatment child had fever, loose motions, vomitings, neutropenia on various occasions which was treated with supportive care including IV antibiotics, IV fluids, and blood components therapy. The patient recovered and was discharged after through monitoring. The patient was on regular follow-up and is doing well.


Characterization and evaluation of MLL gene abnormalities other than translocations may be very important in understanding the leukemogenic process, and more cases are needed to extend the knowledge about MLL gene aberration mechanisms. It can be speculated that a gene or genes in the 11q23 region might have a role in the regulation of hematopoiesis. In conclusion, further evaluation of the t(2;11)(p21;q23) depends on molecular clarification; at present its pathogenesis significance cannot be defined. Our study confirmed FISH as the most suitable assay for detecting MLL rearrangements because of its sensitivity and speed, and it should be included into battery of investigations while treating childhood acute leukemia.


The authors acknowledge the parents of the child for accepting to give the consent and the management of Apollo Hospitals for their support.


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