Minimal Residual Disease Detection by Real Time PCR of the Dominant-Negative Isoform of Ikaros in Patients with Acute Lymphoblastic Leukemia.

Introduction
 The transcription factor Ikaros plays an important role in lymphoid development and proliferation. We and other groups have demonstrated overexpression of the dominant-negative isoform of Ikaros, Ik-6, in patients with acute lymphoblastic leukemia (ALL). By alternative splicing Ik-6 lacks exon3-6, and the junction of exon2 and 7 is a specific sequence for Ik-6. Using this leukemia-specific sequence as a molecular marker, we monitored minimal residual disease (MRD) of ALL patients.

Patients and methods
 We designed a Taqman probe on the junction of exon2 and 7 of Ik-6. Consecutive real time PCR was performed on Ik-6 positve ALL patients. Ik-6 copy numbers were expressed as a ratio to copy numbers of the control gene GAPDH.

Results
 Patient 1
 This 27-year-old patient was found to have ALL with normal karyotype and treated with induction chemotherapy. Unfortunately, her disease was primary refractory and then she received peripheral blood stem cell transplantation (PBSCT) from HLA-identical sibling donor and discharged hospital at day 72 after PBSCT with CR. She was readmitted 10 months later because of recurrence of ALL. After repeating donor leukocyte infusion and chemotherapy, she could not reach CR and received second bone marrow transplantation (BMT) from unrelated donor. Her disease remained CR for one year, however real time PCR of Ik-6 could not remained negative and predicted later relapse.

Patient 2
 The 49-year-old patient was diagnosed with Philadelphia chromosome-positive ALL and achieved CR with induction chemotherapy. She was treated with repeated consolidation chemotherapy and her disease remained CR for 7 months, however real time PCR of Ik-6 became positive and predicted later relapse.

Patient 3
 ALL with normal karyotype was diagnosed in this 39-year-old patient. He received induction chemotherapy and achieved CR. Despite repeated consolidation chemotherapy, his disease recurred after 5 months. He was refered to our hospital for PBSCT from HLA-identical sibling donor, however, even after PBSCT, real time PCR of Ik-6 remained positive and predicted later relapse.

Patient 4
 This 64-year-old patient was diagnosed with ALL with normal karyotype. She was treated with induction chemotherapy and achived CR. Her disease remained CR with consolidation and maintenance chemotherapy for one year, and real time PCR of Ik-6 is negative all the time.

Patient
 5 ALL with Philadelphia chromosome was diagnosed in this 16-year-old patient. She achieved CR with induction chemotherapy and was refered to our hospital for BMT from HLA-identical sibling donor. She discharged our hospital at day 39 after BMT with CR and minor BCR/ABL transcript was not detectable. One year later thrombocytopenia recurred and she was maintained on Imatinib without reaching CR. After 6 months she received second BMT from unrelated donor and discharged our hospital at day 77 with CR. At discharge, real time PCR of Ik-6 was negative and minor BCR/ABL transcript was not detectable.

Discussion
 Our results demonstrate that real time PCR of Ik-6 is useful to monitor MRD in patients with Ik-6 positive ALL.