A 6-year-old boy was referred to a cancer hospital in August 2012 with chief complaints of low grade fever, petechiae, ecchymosis and bone pain. No evidence of gum hypertrophy or bleeding was noticed at presentation. His complete blood count revealed an anemic condition, with decreased hemoglobin level of 4.5 gm/dL; platelet count 13,000/mm³; and total white blood cell count 7,100 /mm³. Further hematological examination revealed hyper cellular bone marrow with 90% blasts while peripheral blood showed 35% blasts cells. Other clinical finding included presence of lymphadenopathy. Flowcytometric immunophenotyping performed on peripheral blood demonstrated predominant lymphoid blasts cells showing percent positivity as follows: B-Lymphoid markers: CD10-94%; CD19-96%; CD20-16%; CD10/CD19 (co-expression)-94%; CD22-92%; Leucocytic common antigen: CD45-99%; non lineage specific markers:CD34-3%; HLA-DR-97%; myeloid markers: CD13-0%; CD33-0%; CD117-3%; and T-lymphoid markers: CD3-1%, CD5-0% and CD7-0%. These combined pathologic findings from the hematological, bone marrow, and immunophenotyping were suggestive of ALL, and the patient was finally diagnosed with Precursor-B-ALL subtype.

Conventional cytogenetic analysis was performed on bone marrow aspirate withdrawn at diagnosis as per standard unstimulated direct (0 h, 3 h) and short term (24 h, 48 h) cell culture as per previous reports 5. In brief, cells were cultured in RPMI (Sigma, Schnelldorf, Germany) medium supplemented with 20% FBS (Gibco, Grand Island, NY) at 37°C. After incubation at respective time intervals, the cells were harvested i.e. cell-growth was arrested by exposure to colchicine (HiMedia, 4mg/ml) for 45 minutes, then hypotonised by 0.075 M KCl for 20 minutes, then fixed with chilled Carnoy s fixative (methanol: glacial acetic acid 3:1). Fixed cells were dropped on chilled frosted slides, then aged overnight at 60°C and stained for GTG banding at resolution of about 400-band level. At least 25 metaphase plates were screened, and 4-5 well spread metaphases were photographed and karyotyped using Ikaros Metasystem Software (Gmbh, Germany). The karyotype was interpreted according to ISCN 2009 6.

The FISH analysis was performed from cultured bone marrow cells on metaphase cells using a Vysis LSI probe in order to understand the probable breakpoints involved in this novel rearrangement. The LSI probe was labeled with Spectrum orange specific for 15q22 locus (Abbott Molecular, Des Plaines, IL). The slides were hybridized overnight according to the Vysis protocol and analyzed using Isis Software (Gmbh, Germany).

In order to rule out the presence of any cryptic, recurrent chromosomal rearrangements such as BCR-ABL, ETV6-RUNX1, TCF3-PBX1, and MLL-AFF1, we did multiplex nested reverse transcriptase PCR as per previous report with minor modification 7. Briefly, total RNA was extracted from the bone marrow aspirate using QIAamp RNA blood mini kit (Qiagen, Hilden, Germany). Fifty nanograms of total RNA were used to prepare cDNA using random hexamer primer (Fermentas, Hanover, MD) and Sensiscript RT Kit (Qiagen). This was followed by two rounds of nested RT-PCR for detection analysis of the fusion genes. Appropriate internal control was also analysed to check the integrity of RNA.

Cytogenetic investigations of the bone marrow cells revealed an abnormal karyotype with novel chromosomal rearrangements involving chromosomes 10 and 15, resulting in the karyotype 46,XY,t(10;15)(q22;q22) pattern in all the cells analysed Figure 1. Microscopic evaluation of the metaphase chromosomes revealed reciprocal translocation of a chromosomal segment from the long arm of chromosome 10q22 to the long arm of 15q22, while 15q22 chromosomal segment was translocated to 10q22 thereby demonstrating a novel translocation t(10;15)(q22;q22). Standard phytohemagglutinin-stimulated peripheral blood lymphocyte culture revealed a normal male karyotype, thereby ruling out the possibility of a germ-line origin for this translocation.

The result of metaphase FISH analysis using the LSI Vysis probe for 15q22 locus is shown in Figure 1. FISH analysis on metaphase cells revealed two orange signals specific to 15q22 locus signal pattern in all the metaphases. Interestingly, out of the two orange signals, one orange signal was located on the long arm of chromosome 10q22 instead of chromosome 15q22 signal absent on the der(15), while the other orange signal was located on normal chromosome 15 thereby confirming presence of rearrangement between 10q22 and 15q22.

RT-PCR analysis showed no amplification for the four selected gene fusion transcripts, thereby ruling out the presence of any cryptic rearrangements. Amplification of the internal control was successful suggesting the presence of intact RNA.

The diagnosis of ALL is based on cytomorphology, immunophenotyping, cytogenetic and molecular genetic analysis of the leukemic blast cells in the peripheral blood and bone marrow 8. Analysis of metaphase chromosomes by G-banding is still the gold standard and best method to evaluate chromosomal abnormalities as it provides a global overview of the genome and a baseline to trace the evolution of the disease. The recognition of common genetic alterations in leukemic cells has contributed greatly to our understanding of the pathogenesis and prognosis of ALL 3. The most common prognostic fusion oncogenes in pediatric ALL include BCR-ABL, ETV6-RUNX1, MLL-AF4 and TCF3-PBX1 9. Besides a substantial portion of ALL patients lacking common translocations are characterized by change in their ploidy status (like hypodiploidy, hyperdiploidy etc.], which also serves as an important prognostic factor 1011. The observation that up to 25% of pediatric ALL cases lack a recurring chromosomal alteration and the identification of translocation-encoded fusions at birth or in years prior to the onset of leukemia 12 indicates that additional genetic alterations cooperate in leukaemogenesis.

Reported here is a pediatric case of precursor-B-ALL with a novel chromosomal rearrangement t(10;15)(q22;q22) that has not yet been reported in any literature. The initial diagnosis of ALL was made on the basis of clinical features at presentation, high bone marrow cellularity, elevated blast cells in marrow and peripheral blood, low hemoglobin and platelet counts. Flowcytometric immunophenotyping demonstrated predominant lymphoid blasts cells that lacked any myeloid or T-lymphoid markers, while they expressed predominantly B-Lymphoid (CD10, CD19, CD22, HLA-DR and co-expression of CD10 & CD19) markers, which led to the final diagnosis of Precursor- B-ALL subtype. In the present case, we were able to detect t(10;15)(q22;q22) by using a standard cytogenetic technique at 400 band resolution. Microscopic evaluation of leukemic cells revealed that chromosomal segment from the long arm of chromosome 15q22 region was translocated to the distal end on the long arm of chromosome 10q22, resulting in the abnormal karyotype 46,XY,t(10;15)(q22;q22) in all the cells analyzed. Since this abnormality was detected in all the cells, standard phytohemagglutinin-stimulated peripheral blood lymphocyte culture revealed a normal male karyotype, thereby ruling out the possibility of a germ-line origin of this translocation. In order to further understand the breakpoint involvement, we did FISH analysis on metaphase slides using probes located at 15q22. On the basis of FISH results we could ascertain that 15q22 was involved in this rearrangement further confirming our chromosomal findings.

This type of novel chromosomal translocation may entail rearrangements of certain cancer associated tumor suppressor gene/(s) involved in apoptosis and transcription regulation, whose alterations may lead to loss of their normal functions and activation of oncogenic pathways leading to leukemogenesis. As for the actual mechanism or the genes involved in this rearrangement, it still remains unclear. Thus, further molecular characterization of this translocation can unravel the molecular mechanism for disease pathogenesis and will provide further insights into the significance of various gene alterations thereby development of novel targeted therapies.