Quantitative Monitoring Of Wilms’ Tumor 1 Expression In Predicting Relapse After Allogeneic Hematopoietic Stem Cell Transplantation In Patients With Acute Leukemia and Myelodysplastic Syndrome

Relapse is the major cause of treatment failure after allogeneic hematopoietic cell transplantation (HCT) for leukemia and myelodysplastic syndrome (MDS). In order to improve the outcome of leukemia patients after allogeneic HCT, it is imperative to identify reliable markers to predict impending relapse. Wilms’ tumor antigen (WT1) is overexpressed in the majority of leukemia and MDS patients and is being considered as a possible universal diagnostic marker for minimal residual disease (MRD), especially since no chromosomal translocation has nearly the frequency of association as WT1 does with leukemia.

In this study we prospectively evaluated the prognostic value of MRD monitoring by qRT-PCR for WT1 transcripts. WT1 transcript levels in peripheral blood mononuclear cells (PBMC) were measured utilizing SYBR-Green qRT-PCR on the ABI7300 instrument (Applied Biosystems, Carlsbad, CA) and results were expressed as a ratio of WT1/c-ABL transcript copies normalized by 10⁴ (WT1 ratio: WT1/c-ABLx10⁴). PBMC samples were obtained monthly for 6 months post-HCT, then on alternating monthly schedule until 3 years post-HCT. Patients >18y.o. with confirmed diagnosis of MDS with <20% blasts, AML/ALL in 1^st or 2^ndCR, and CML in chronic phase undergoing HCT were eligible for the study.

A total of 83 patients (median age: 54, range: 19-74) with AML (n=39), ALL (n=24), MDS (n=17), or CML (n=3) received allogeneic HCT after fully ablative (n=39) or reduced-intensity (n=44) conditioning. Donor sources were matched related (n=33), unrelated (n=50), or umbilical cord blood (n=2). Fifty-one patients were considered low-risk (AML/ALL in CR1, CML in 1^stchronic phase, or MDS-RA/RARS subtypes) while the remaining 32 patients were considered high risk.

Sixteen of 83 patients relapsed with a median time of 238 days post-HCT (range: 76-747). The minimum WT1 ratio that gave specificity of 100% in predicting relapse was 50 (95% binomial exact CI: 92.5-100%), as none of the non-relapsed patients crossed this level. Of 16 patients who relapsed, 12 crossed the WT1 ratio of 50, providing a sensitivity of 75% (95% binomial exact CI: 48- 93%). The positive predictive value (PPV) and the negative predictive value (NPV) performance parameters for the WT1 ratio of 50 were 100% and 94.4%, respectively (Table 1). There was an average number of 63 days (SD=29.3) from crossing the WT1 ratio of 50 to hematologic relapse for the 12 relapsed patients. Since PBMC samples from healthy donors consistently demonstrated a WT1 ratio <10, we also examined different WT1 cutoff ratios (10 to 50) for their performance characteristics (Table 1). Compared with the WT1 ratio of 50, a cutoff ratio at 20 resulted in an increased sensitivity (87.5%) for relapse prediction and days to relapse (78 days), while the specificity and PPV decreased to 85% and 58.3%, respectively. The performance of the cutoff ratio of 20 was improved on PPV (69%) and days to relapse (85 days) in a subgroup of patients with high-risk disease while maintaining good sensitivity and specificity above 80%. Univariate analysis showed WT1 ratios (as a continuous variable), crossing the WT1 ratio of 20 (as a time-dependent variable), high risk disease, and donor age were significantly associated with relapse. In multivariate analysis, crossing the WT1 ratio of 20 remained the only significant factor predicting relapse (HR 56.9 [18-189], p<0.0001).

In summary, our data demonstrate that the quantitative measurement of WT1 transcripts is a reliable marker to assess MRD post-HCT for leukemia patients, and its real-time prospective monitoring provides a 2-3 month window of opportunity to introduce medical/immunologic interventions prior to overt hematologic relapse.