Quantitation of WT1 Transcripts by Copy Number Provides a Sensitive Indication of Minimal Residual Disease (MRD) and Response to Adoptive Cell Therapy in Patients Transplanted for Ph(+) CML.

Abstract 4489

The Wilm's tumor gene, WT-1, is markedly overexpressed in a high proportion of acute leukemias, advanced MDS and Ph(+) CML. Prior studies, employing semiquantitative PCR-based techniques, have suggested that elevated levels of WT-1 transcripts in bone marrow and PBMC are indicative of MRD, and predictive of relapse in AML and CML following chemotherapy. We have now developed a quantitative real time PCR-based assay for WT-1 transcripts and used it to evaluate the long term effects of donor lymphocyte infusions in a series of patients with Ph(+) CML treated for relapse or persistent MRD following allogeneic marrow transplants between 1993 and 2004 who are currently in molecular or cytogenetic remission.

To quantitate WT-1 mRNA copy number in the marrow and PBMC, total RNA from patient samples received at different times that had been extracted with trizol reagent and stored at -20°C was transcribed into cDNA using MMLV reverse transcriptase. Real time PCR was performed with 1-2 μg of total RNA using the ABI 7300 instrument with a 25 base pair FAM labeled probe containing a sequence located at the boundary of exon 6 and 7 of the WT-1 gene in order to eliminate amplification of genomic DNA and insure measurement of all transcribed WT-1 isoforms. The forward and reverse primers were synthesized from sequences contained in exon 6 and 7 of the WT-1 gene respectively. Cloned plasmids containing defined copy numbers of WT-1 and human GAPDH were used to prepare standard curves using serial dilutions. Quantitation of WT-1 copy number expression was performed by calibration against these standard curves. The expression of WT-1 was normalized against the expression of the control GAPDH to adjust for variation in RNA and cDNA synthesis.

Using this assay, we evaluated sequential samples from each patient for correlations between WT-1 copy number detected and the results of qualitative PCR assays for bcr/abl fusion gene transcripts previously performed on the same samples using nested primers as well as the results of clinical, hematologic and cytogenetic analyses of each patient obtained at each time of sampling. Blood samples from a group of 8 normal volunteers served as negative controls.

The average WT-1 copy number in the blood of normal volunteers was 25 copies/ μg RNA (range 2-60). With one exception, blood and marrow samples from patients with CML in molecular remission (bcr/abl not detected) had WT-1 copy numbers < 50 copies/μg RNA. One sample of marrow from a patient who had been bcr/abl ⁺ until 6 months post transplant and had WT-1 copy numbers that had fallen from 12,932 one month post transplant to 1855 at that time, became bcr/abl negative at 11 months but still had a WT-1 copy number of 931/ μg RNA. In contrast, patients with molecular disease (+ bcr/abl) maintained WT-1 copy numbers ranging from 70-1000/ μg RNA. Samples from patients with cytogenetic relapse consistently had WT-1 copy numbers exceeding 1000/ μg RNA, with levels exceeding 10,000 in 4/6 such patients tested. In a subset of patients who have failed to achieve or sustain molecular remission following DLI, marrow and blood samples with detectable bcr/abl transcripts and WT-1 copy numbers ranging from 60-3000 have been repeatedly detected for periods of up to 6 years without additional treatment and without disease progression implying an ongoing mechanism of disease control. Therefore, quantitation of WT-1 copy number provides a useful technique for monitoring minimal residual disease and may be helpful in identification of patients at higher risk of cytogenetic relapse.