Simultaneous Sessions I: Modeling and therapeutic targeting in acute lymphoblastic leukemia I
Alterations of the tumor suppressor gene TP53 are observed in 15-20% of patients with acute lymphoid leukemia (ALL) and are associated with resistance to standard treatment regimens and inferior survival. TP53 mutations (TP53mt) have been reported as an early leukemogenic event in patients with acute myeloid leukemia. In ALL however, only limited data are available.
Study the pre-leukemic mutation pattern of TP53 gene in ALL patients. Therefore, we analyzed the kinetics of the TP53mt in bone marrow and peripheral blood in the group of 43 adult ALL patients harboring TP53mt at the diagnosis (Dx) and compared it to the kinetics of standard EuroMRD IG/TR minimal residual disease (MRD). Moreover, TP53mt were assessed in the distinct hematopoietic populations.
Patients received induction/consolidation treatment according to the GMALL 07/03 and GMALL register protocol. For each patient, TP53-mutations were analyzed in at least one Dx/MRD-positive (n = 54) and one MRD-negative sample, if available (n = 41). The presence of TP53mt was validated by Sanger sequencing and quantified using UMI-based next generation amplicon sequencing (sensitivity: 5x10−3). In 5 patients, selected based on TP53mt-status and on the availability of viable cells, distinct hematopoietic populations were sorted using FACS Aria sorter in an MRD-negative follow-up sample and analyzed for the presence of the TP53mt using allele-specific digital droplet PCR (ddPCR, sensitivity: 1,5x10−3-1x10−2). MRD was monitored using real-time quantitative PCR of clonal immune gene rearrangements (sensitivity 1x10−5) and multiparameter flow cytometry.
In our series of 43 TP53mt ALL patients (B-ALL 90.7%, T-ALL 9.3%) MRD analysis revealed quantifiable IG/TR MRD persistence in 7 patients (16.3%), complete MRD remission in 30 patients (69.8%), and MRD persistence at not quantifiable levels in 6 patients (14.0%). In 9 of the MRD-negative patients (20.9%) the TP53-mutation was detected at levels between 2.0 and 17.4% (median 8.1%), pointing to the presence of TP53mt in a pre-leukemic compartment. In 5 of 7 MRD-persisters also TP53 mutations were detected. In the 2 remaining patients MRD-level was below the TP53-assay sensitivity. ddPCR analysis of the sorted, progenitor and mature hematopoietic populations revealed the presence of TP53mt in early hematopoietic cells including multipotent progenitors (MPP: CD19-34+38−), common myeloid/lymphoid progenitors (CD19-34+38+13/33−) and early myeloid progenitors (CD34+19-38+13/33+) but also in differentiated cells as granulocytes (SSChighCD13/33dim) and monocytes (CD19-34-3-13/33high). TP53mt was generally absent in the mature T cells but present in the B-cell precursors defined as CD19+10+20-34+ cells (Figure 1); altogether this data point to the pre-leukemic origin of TP53mt. Interestingly, one patient who relapsed after two years with a fully unrelated IG/TR rearrangement still harbored the TP53mt from Dx sample, pointing to the involvement of the TP53mt in the ALL reoccurrence. The detailed analysis of other relapse-associated mutations is currently ongoing. The correlation analysis of clinical outcome with the observed TP53 mutation pattern will also be performed.
Hereby, we have shown that in a considerable number of adult ALL patients a TP53 mutation is present in pre-leukemic cells, causing clonal expansions in different hematopoietic compartments, and thus persisting in MRD negative follow-up samples. Considering TP53 somatic mutations as an MRD-marker requires careful interpretation.