Development of an allele-specific minimal residual disease assay for patients with juvenile myelomonocytic leukemia

Abstract

Juvenile myelomonocytic leukemia is an aggressive and frequently lethal myeloproliferative disorder of childhood. Somatic mutations in NRAS, KRAS, or PTPN11 occur in 60% of cases. Monitoring disease status is difficult because of the lack of characteristic leukemic blasts at diagnosis. We designed a fluorescently based, allele-specific polymerase chain reaction assay called TaqMAMA to detect the most common RAS or PTPN11 mutations. We analyzed peripheral blood and/or bone marrow of 25 patients for levels of mutant alleles over time. Analysis of pre-hematopoietic stem-cell transplantation, samples revealed a broad distribution of the quantity of the mutant alleles. After hematopoietic stem-cell transplantation, the level of the mutant allele rose rapidly in patients who relapsed and correlated well with falling donor chimerism. Simultaneously ana- IntroductionJuvenile myelomonocytic leukemia (JMML) is an aggressive clonal malignancy of young children characterized by overproduction of myeloid lineage cells that infiltrate hematopoietic and nonhematopoietic tissues. 1,2 More than 70% of patients have a mutation in the NF1, RAS, or PTPN11 genes, which encode proteins involved in Ras signaling. 3 Animal models demonstrate that Nf1 Ϫ/Ϫ , Kras G12D , and Ptpn11 D61G mutant mice all develop fatal myeloproliferative disorders that model JMML in vivo and in vitro. [4][5][6][7][8] These observations strongly support the role of hyperactive Ras in initiating JMML.The diagnosis and treatment of patients present great challenges to physicians. Currently, the diagnosis is made by meeting a constellation of clinical and laboratory criteria, 1,9 including the demonstration of fewer than 20% blasts in the bone marrow (BM). Historically, patients with JMML treated with conventional chemotherapy have an event-free survival of less than 15%. 9,10 Allogeneic hematopoietic stem cell transplantation (HSCT) will cure up to 50% of patients. 11 Novel treatment approaches are urgently needed for this disease, given the dismal survival with traditional chemotherapy and the severity of late effects that HSCT confers on these young children.However, defining response to therapy is another major challenge for clinicians. 12 Current response criteria are broadly defined as a decreasing white blood cell count and lessening organomegaly. Detecting minimal residual disease (MRD) in JMML relies on tracking donor chimerism after HSCT. One quantitative donor chimerism tracking method used and reported in JMML involves polymerase chain reaction (PCR) amplification of short tandem repeats with comparison of informative loci. 13 Thus, delivering chemotherapy or novel agents before HSCT is severely limited by our inability to follow disease burden in these patients.Allele-specific oligonucleotide-PCR has been developed to detect single nucleotide substitution levels in various cancers 14 and involves designing primers that will preferentially amplify the mutant allele over the wild-type (WT) allele by exploiting differences in the amplification efficie...

RAS Mutations Contribute to Evolution of Chronic Myelomonocytic Leukemia to the Proliferative Variant

“…To this aim, we conducted a longitudinal monitoring of chronic myelomonocytic leukemia course first by sequencing and then by allele-specific PCR. In fact, among the various assays developed for early detection of specific mutations, tracking of disease progression, and evaluation of molecular response to therapy, which are crucial issues in clinical practice, allele-specific PCR represents a simple, fast, and sensitive method (35,36). Direct sequencing detected a significantly higher incidence of RAS mutations in MP-CMML patients, consistent with previous reports (1,5,(7)(8)(9)14).…”

Childhood Myelodysplastic Syndrome: Focus on the Approach to Diagnosis and Treatment of Juvenile Myelomonocytic Leukemia

“…In addition, novel methods of detecting relapse early post-transplant beyond traditional donor chimerism include sorted cell donor chimerism, quantitative PCR to assess chimerism, as well as allele-specific PCR strategies to detect mutation-specific clones. 42,46 Such sensitive measurements may facilitate earlier and more rapid withdrawal of immunosuppression or the initiation of novel targeted therapies. There are minimal data on the utility of donor lymphocyte infusions.…”

“…41 In addition to such criteria, allele-specific, polymerase chain reaction (PCR)-based strategies have been developed to track specific mutations in patients on therapy and will be tested in future clinical trials to determine their role prospectively. 42 Future targeted therapies are being intensely investigated in a variety of laboratories around the country. Such potential therapies include Janus kinase (JAK) inhibitors, based on work demonstrating a pSTAT5 response in JMML cells upon exposure to subtherapeutic concentrations of GM-CSF, 43 as well as other signal transduction components of Ras signaling, such as PI3K (phosphoinositide 3-kinase), MEK (MAPK/extracellular-signal-regulated kinase [ERK] kinase), and mTOR (mammalian target of rapamycin) inhibitors.…”

Aberrant GM-CSF signal transduction pathway in juvenile myelomonocytic leukemia assayed by flow cytometric intracellular STAT5 phosphorylation measurement

“…Using this method, the authors demonstrated the possibility to quantify disease burden before HSCT and to measure minimal residual disease after HSCT in JMML patients. 12 In this context, upon confirmation and extension in larger series, flow cytometric monitoring of STAT5 phosphorylation signature may also represent a rapid and sensitive tool to evaluate response to therapy even in those patients without PTPN11 and RAS mutations.…”