A case of acute myeloid leukemia-M 2 with trisomy 4 in addition to t ( 8 ; 21 )

chronic myelomonocytic leukemia (CMMoL), and one with unclassiÞ ed preleukemia. The coincidence of +4 with t(8;21) or its variant t(6;21;8) has been observed in at least two cases of ANLL (M1 and M2), is therefore recurrent. Apparently, trisomy 4 has no prognostic sifgniÞ cance in ANLL; with the exception of the cases bearing c-kit mutations that are associated with a rapid disease progression. Trisomy 4 has been described in two cases of T-cell acute lymphoblastic leukemia as the sole chromosomal anomaly. Combined trisomies of chromosomes 4 and 10 are reported in children with B-progenitor cell acute lymphocytic leukemia and has shown a favorable prognostic association. Patients with chromosomes 4 or 10 trisomies as a sole anomaly have an extremely favorable 44-year event free survival after antimetabolite-based chemotherapy.[4]


Introduction
t(8;21)(q22;q22) is the most frequently observed karyotypic abnormality associated with acute myeloid leukemia (AML), speciÞ cally in FAB M2.Trisomy 4 as the sole anomaly is a rare chromosomal abnormality associated with a speciÞ c subtype of primary acute non lymphocytic leukemia (ANLL) and secondary (treatmentrelated) ANLL with myelomonocytic morphology; it has been found with the same frequencies in the M1, M2, and M4 FAB phenotypes. [1,2]A retrospective analysis on the clinical and laboratory data of 21 cases of acute leukemia (AL) with trisomy 4 was performed by Pan et al. and showed that AL with trisomy 4 have unique clinical and laboratory features and a poor prognosis.
M2 was the most frequent subtype in this series (9 out of 21 cases). [3]Association of +4 with double minute chromosomes has been described in 10 cases; 5 with AML-M2, 2 with AML-M4, 1 with refractory anemia with excess of blasts in transformation (RAEB-T), one with M:E ratio was altered, and megakaryocytes were not seen, lymphocytes 8%, eosinophills 1%, polymorphs 3%, band cells 2%, metamyelocytes 1%, myelocyte 8%, promyelocytes 3%, and blast cells 72%.Final diagnosis based on morphological and cytochemistry Þ ndings was AML with M2 subtype as per French-American-British classiÞ cation.Constitutional nature of trisomy 4 could not be ruled out.After 1 month of sample received, the patient was lost to follow up.

Chromosome preparation
A G-banded chromosome study was performed using standard cytogenetic protocol.Briefly, unstimulated cultures of bone marrow aspirate were set up in RPMI-1640 medium supplemented with 20% newborn calf serum, l-glutamine, and antibiotics (penicillin and streptomycin).The cells were cultured for 24 and 48 h in 5% CO 2 incubator.Following overnight incubation in presence of Colcemid (10 µL/8 mL of culture) the cultures were exposed to hypotonic solution (0.075 mol/L KCl) and Þ xed with methanol:acetic acid (3:1).The slides were prepared by air-dry method and stained with GTG-banding.Twenty metaphases were analyzed and karyograms were prepared using the Cytovision computer-assisted karyotyping system (Applied Imaging, NewCastle Upon Tyne, UK).The karyotypes were described according to the International System for Human Cytogenetics Nomenclature 2005. [5]uorescence in situ hybridization (FISH) assay

Chromosome analysis
Classical chromosome analysis detected an abnormal female chromosome complement.Karyotyping results revealed trisomy of 4 with t(8;21) in all metaphase plates in 15 metaphases [Figure 1].

FISH analysis
The interphase and metaphase FISH results were one green, one orange, and two green-orange or yellow  found in association with AML.The Mitelman database for chromosomal aberrations in cancer queried for trisomy 4 with t(8;21) in AML showed only two cases. [6]ins or losses of chromosomes are frequent Þ ndings in AML, more common being monosomy 7 and trisomy oncogenes ampliÞ cation, and leukemogeneisis has yet to be established. [7,8]r patient was lost to follow up hence the CD56 status was unclear.Therefore, relation between CD56 expression and trisomy 4 needs further investigation.While development of AML with trisomy 4 secondary to chemo or radiotherapy has also been suggested, our patient had no history of long-term medication, radiotherapy, or any relevant occupational exposure.In conclusion, based on the morphological, cytochemistry, and clinical features the present case of AML-M2 is a rare case in terms of cytogenetic results.Even though trisomy 4 is likely to be a secondary event after t(8;21) translocation, the presence of this additional numberical aberration may deÞ ne a distinctive subtype.Follow up of more such cases over a period of time is required to know the possible prognostic effect of this cytogenetic entity. [9] Address forCorrespondence: Sonal R. Bakshi, Cell Biology Division, Department of Cancer Biology, G. C. and R.I., NCH Campus, Asarwa, Ahmedabad -380 016, India.E-mail: cbdgcri@rediffmail.comIndian Journal of Human Genetics January-April 2008 Volume 14 Issue 1 20

FISH
procedure was performed on interphase and metaphase cells following the manufacturer's (Abbott Molecular, Inc., Des Plaines, IL, USA) guidelines.The LSI AML-ETO dual-color dual-fusion probe was used to determine the AML-ETO fusion status to conÞ rm the diagnosis of AML-M2 subtype.Whole chromosome paint for chromosome 4 with spectrum orange was applied to conÞ rm trisomy and/or cryptic rearrangements of chromosome 4.
fusion signals indicating AML-ETO fusion positive sample [Figure 2A].The whole chromosome paint FISH conÞ rmed trisomy of chromosome 4 with no other cryptic rearrangements [Figure 2B].Discussion Trisomy 4 is a rare nonrandom cytogenetic abnormality

8 .
Most of such changes are not restricted to any speciÞ c FAB types of AML, are often also associated with secondary AML and AML with pre-existing myelodysplasia, or during clonal progression of AML.Rarely gain of chromosome 4 or chromosomes 10 are reported as the sole abnormality in AML.Some morphologic subtypes of AML are associated with speciÞ c chromosomal abnormalities.Most of these abnormalities are chromosomal translocations which amplify or activate chimeric genes situated near the breakpoints of translocations.Trisomy 4 occurs in AML with frequency of <1% and a strong association with the presence of double minutes has been described.Double minutes were not observed in our patient.CD56 expression in AML is reported in granulocytic sarcoma and multidrug resistance, and is known to confer poor prognosis in AML-M2 with t(8;21) and acute promyelocytic leukemia.It has been suggested that there might exist a dosage effect of certain genes resulting in growth advantage of malignant cells.In the case of AML with trisomy 4 and double minutes, V-myc myelocytomatosis viral oncogene homolog (avian) ampliÞ cation is a common Þ nding.Jennings et al, have also suggested that acquisition of trisomy 8 leading to MYC locus ampliÞ cation might underline the molecular mechanisms for the clonal progression of chronic myeloid leukemia.A direct link between chromosomal gain,