Development of an Allele-Specific Minimal Residual Disease Assay for Patients with Juvenile Myelomonocytic Leukemia-Moving beyond Clinical Assessment.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of childhood characterized by a high frequency of somatic mutations in NRAS, KRAS2, or PTPN11 genes as well as LOH of the NF1 allele (mainly in patients with type 1 neurofibromatosis). Activation of the Ras pathway is the biochemical consequence of these lesions. Hematopoietic stem cell transplantation (HSCT) is the only known curative treatment, with event-free survival (EFS) rates approaching 50–60%. Leukemia relapse still remains the main cause of treatment failure. Monitoring minimal residual disease (MRD) before and after HSCT is challenging due to the lack of tractable molecular markers. We designed and validated a fluorescently based, allele-specific polymerase chain reaction assay called TaqMAMA that can detect point mutations in eight of the most common RAS or PTPN11 mutations with a sensitivity of 1/1000 copies of the wildtype allele. We analyzed the peripheral blood (PB) and/or bone marrow (BM) in 22 patients for the levels of these mutant alleles at diagnosis and pre-HSCT. Eighteen of these patients had weekly-monthly PB and/or BM samples available after 21 HSCTs (3 children had second transplants). We compared the percentage of mutant DNA with rising levels of autologous cells measured by chimerism analysis after HSCT, which is the current surrogate marker used to detect relapse. Analysis of patients at initial diagnosis and pre-HSCT revealed a broad distribution of the frequency of the mutant alleles, regardless of the use or intensity of pre-HSCT chemotherapy. As shown in the table, levels of disease burden prior to HSCT were not predictive of relapse. After HSCT, the level of the mutant allele rose rapidly in patients who relapsed and correlated well with analysis of chimerism (r=0.86, p<0.01), thus validating the assay. In addition, the concordance rate between 15 simultaneously obtained PB and BM samples was excellent in 13 of the pairs, with a maximum of a 3.7 fold difference at < 1% of the mutant allele. In conclusion, patients with JMML harboring the most common Ras pathway mutations can now be monitored using an allele specific MRD assay. Most importantly, these assays can measure the frequency of mutant alleles in PB and provide a sensitive new strategy for MRD monitoring that can evaluate the molecular response to new therapeutic strategies for JMML.