A 17-year-old boy was admitted in Tata Memorial Hospital with complaints of anemia, vomiting, and distension of the abdomen. His hematological findings were as follows: WBC 23 × 109/l, platelets 112 × 109/l, and Hb 7.66 gms/dl. Bone marrow aspirate (BMA) showed 90% blasts with cytoplasmic granules and Auer rods. The patient was diagnosed as AML with FAB M1 subtype. Immunophenotype screening of blasts by flow cytometry showed positive CD13 69%, CD33 74%, CD34 27%, and HLA DR 82%; it showed negative CD14, CD 36, as well as B- and T-cell markers.
Conventional cytogenetics was carried out on unstimulated bone marrow cells. The BMA cells were harvested after 30 min and/or 24 h of incubation. Cytogenetic analysis at diagnosis revealed normal karyotypes as well as abnormal karyotypes . Abnormal karyotype was defined as 46,XY,dic(5)(q22). Loss of Y was detected as a random aberration [, ]. VNTR study using markers D17S250 and D17S579 was performed for chimerism analysis of post-transplanted recipient marrow. The PCR product length of donor and recipient DNA revealed polymorphism . The patient was immediately put on primary HDCT. HDCT, consisting of idarubicin 200 mg/m2 for 3 days and cytaribine 100 mg/m2 for 7 days, was administered. Thereafter, he was reinduced with cytaribine 20 gm/m2 for 5 days followed by consolidation of Ara-C for 4 days in two courses. Hematopathology of post-chemotherapy BMA (3 months after HDCT) showed 6% blasts. Cytogenetic analysis revealed a major clone of normal karyotypes, 46,XY (13 cells) and a minor clone of 46,XY,dic(5q) (2 cells) . Four months after diagnosis the patient underwent allogeneic peripheral stem cell transplantation from his HLA-matched donor brother. Before transplantation the patient received a myeloablative regimen which included busulphan 16 mg/kg for 4 days and cyclophosphamide 128 mg/kg for 2 days. The patient also received GVHD (graft vs. host disease) prophylaxis with cyclosporin, methotrexate (600 mg/m2), and GM-CSF (5 µg/kg). On day 7 of transplantation, he developed grade I skin GVHD and upper GI GVHD. He was treated with methylprednisolone for 7 days. His bone marrow examination showed hypocellular marrow with 1% blasts. Cytogenetic evaluation on the 28th day after PBSCT showed a normal karyotype, 46,XY . Thereafter, the patient remained in remission for 4½ months. VNTR analysis revealed donor chimerism . A month later, the patient presented to the clinic with fever, cough, and splenomegaly. Peripheral blood showed WBC 17.4 × 109/l, platelets 41.1 × 109/l, and 30% blasts. Bone marrow examination revealed 38% myeloid blasts by morphology, indicating relapse. Simultaneously, cytogenetic analysis in marrow cells identified a new hyperdiploid clone with the unusual translocations t(6;17)(p23;p11.2) and t(10;19)(q26.1;q13.3), along with trisomy 8 and der(8)dup inv(8)(q23qter) [ and ]. VNTR analysis showed recipient chimerism in the bone marrow cells . FISH was performed on a fixed cytogenetic preparation of bone marrow cells at relapse, using Vysis Inc. CEP 8, 10, 19q subtelomeric probe (TelVysion) and locus-specific MYC break apart rearrangement probe of locus 8q24. Interphase and metaphase FISH analysis revealed trisomy 8  and confirmed t(10;19) . FISH for trisomy 8 and t(10;19) was also done on a fixed cytogenetic preparation of bone marrow cells at diagnosis to rule out the presence of masked clones of +8, t(10;19) that might have been missed. The analysis revealed absence of trisomy 8 and t(10;19) at diagnosis. The patient was treated with standard recommended doses of fluradibine, idarubicine, Ara-C, and Myelotarg for 4 days. However, the patient failed to respond to chemotherapy. His marrow examination showed 66% blasts. Repeat cytogenetics analysis revealed a similar karyotype picture to that noted before: 47, XY, t(6;17), +8, der(8)dup inv(8)(q23qter),t(10;19). The patient underwent a second transplant, i.e., bone marrow transplant from the same HLA-match brother 52 days after the first relapse. He received melphalan 90 mg/m2 as a nonmyeloablative conditioning regimen. He was also given GVHD prophylaxis which included cyclosporin and methotrexate. A month later, after BMT, hematological evaluation of BMA showed hypocellular marrow with no blasts. Cytogenetic analysis also revealed a normal karyotype pattern, 46,XY. However, the patient developed GVHD within a short period. He had febrile neutropenia and flaring of fungal infection. He was given platelet transfusion for 6 weeks. His CT scan showed multiple enhancing regions in the right hemisphere and the cerebellum. The patient died within 4 months of the second transplantation.
Sequential conventional cytogenetic analysis at diagnosis, in remission, and at relapse
Karyotype, 45,X,-Y,dic(5q22) at diagnosis
Figure 2 VNTR analysis by PCR. Tracks are (M) molecular weight marker ØX174, (R) recipient DNA, (D) donor DNA, (PTR1) (1st post-transplant 1st remission marrow), and (PTR2) (1st post-transplant 2nd remission marrow), (PTR3) (1st post-transplant relapse (more ...)
Karyotype, 47, XY,t(6;17)(p23;p11.2), +8,dup inv(8)(q23qter), t(10;19)(q26.1;q13.3) at relapse
Figure 4 (A) FISH with CEP 8 (aqua) and LSI dual-color MYC probe on metaphase cell shows aqua signal of CEP 8 and orange/green signal of MYC at locus 8q24: signal pattern is indicative of trisomy 8. (B) FISH with CEP 10 (orange) and subtelomeric 19q probe (orange) (more ...)