Case history
A 10-year-old female presented to our hospital in October 2000 because of excessive tiredness, epistaxis and weight loss. Examination revealed moderate hepatosplenomegaly, and a blood count showed hemoglobin 102 g/L, white cell count 179 × 109/L, blasts 1%, promyelocytes 8%, myelocytes 10%, metamyelocytes 29%, eosinophils 1%, basophils 7%, bands 16%, polymorphs 26%, lymphocytes 2% and platelets 917 × 109/L. Leukocyte alkaline phosphatase was 11. Bone marrow examination was consistent with chronic phase CML (CML-CP). Cytogenetic studies showed 25/25 cells with 46, XX, t(9;22), t(11;18), der(16), t(16;?) by R-banding technique. Fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR) studies for BCR/ABL fusion gene were positive. She received interferon-alpha (IFN-α) combined with hydroxyurea therapy. Hydroxyurea was discontinued three weeks later, when white cell count decreased to 5.7 × 109/L, and spleen and liver became non-palpable. Treatment with IFN-α was commenced at a dose of 1.5 million-units (MU)/day. BCR/ABL fusion gene remianed positive (90%~100%) by FISH analysis, which was performed once or twice per year from 2001 to 2005. In May 2005, we boosted the dose of IFN-α from 1.5 to 3 MU/day. Unfortunately, the patient failed to tolerate full-dose IFN-α due to leukopenia (1 × 109/L) complicated with fever. We discontinued IFN-α therapy for 3 months. After this, the dose of IFN-α ranged from 1.5 to 3 MU per week according to white cell count. In January 2006, FISH analysis revealed that the patient achieved complete cytogenetic remission (CCR). At this time, IFN-α was stopped, and imatinib mesylate (IM, 400 mg/d) was given instead according to the patient's selection. BCR/ABL fusion gene was detected using FISH analysis of marrow samples in March, May and August 2006.
In October 2006, the patient was admitted to our department again due to sudden onset of overall osteodynia, especially both in lower extremities, sternum and ribs. A blood count showed hemoglobin 102 g/L, white cell count 5.72 × 109/L, myelocytes 1%, bands 14%, polymorphs 24%, monocytes 12%, eosinophils 1%, lymphocytes 48% and platelets 75 × 109/L. Bone marrow smear revealed 95% blasts that expressed CD34, HLA-DR and the lymphoid antigens CD19, CD20 and CD10. The blasts were myeloperoxidase negative by cytochemistry staining, and cytogenetic analysis showed 25/25 cells with 46, XX. Repeat FISH analysis of this sample confirmed 200/200 metaphase cells to be Ph-negative. After receiving two courses of induction chemotherapy consisting of CMOP regimen (cyclophosphamide, mitoxantone, vincristine and prednisone) and FLAG regimen (fludarabine, cytoarabine and granulocyte-cloning stimulating factor), respectively, the patient achieved complete remission (CR). Unfortunately bone marrow aspirate performed four weeks later showed relapse with 67% lymphoblasts. The karyotype was still normal, and BCR/ABL fusion gene was still negative by FISH. The patient was treated palliatively and died of pulmonary invasive fungi infection in June 2007.
Peripheral blood mononuclear cells (PBMC) isolation, RNA isolation and cDNA synthesis
PBMC were isolated by Ficoll-Hypaque gradient centrifugation. RNA was extracted from the PBMC samples according to the manufacturer's recommendations (Trizol, Gibco, USA): The quality of RNA was analyzed in 0.8% agarose gel stained with ethidium bromide. Two μg RNA was reversely transcribed into the first single-strand cDNA with random hexamer primers, using reverse transcriptase, Superscript II Kit (Gibco, USA). The quality of cDNA was confirmed by RT-PCR for β2 microglubin gene amplification.
RT-PCR for TCR Vα and TCR Vβ subfamily amplification
29 sense TCR Vα primers and a single TCR Cα reverse primer, or 24 TCR Vβ sense primers and a single TCR Cβ primer were used in unlabeled PCR for amplification of the TCR Vα and Vβ subfamilies respectively[
23]. Subsequently, a runoff PCR was performed with fluorescent primers labelled at 5' end with the FAM fluorophore (Cα-FAM or Cβ-FAM) purchased from TIB MOLBIOL GmbH, Berlin, Germany. PCR was performed as described by Puisieux I et al and our previous studies[
16,
23,
24]. Aliquots of the cDNA (1 μl) were amplified in 25 μl reactions with one of the 29 Vα primers and a Cα primer or one of 24 Vβ primers and a Cβ primer. The final reaction mixture contained 0.5 μM sense primer and antisense primer, 0.1 mM dNTP, 1.5 mM MgCl
2, 1×PCR buffer and 1.25 U Taq polymerase (Promega, USA). The amplification was performed on a DNA thermal cycler (BioMetra, Germany). After 3 min denaturation at 94°C, 40 PCR cycles were performed, each cycle consisting of 94°C for 1 min, 60°C for 1 min and 72°C for 1 min, and a final 7 min elongation at 72°C. Then, the products were stored at 4°C.