Backtracking Of The Genetic Events Preceding Acute Leukemia Diagnosis
Authors: Zimmermannova Olga , Zaliova Marketa , Mejstrikova Ester , Fronkova Eva , Jarosova Marie , Zemanova Zuzana , Lizcova Libuse , Al-Shehhi Halima A , Moorman Anthony V. , Vora Ajay J , Meyer Claus , Marschalek Rolf , Stary Jan , Trka Jan , Hrusak Ondrej , Zuna Jan 
Published: October 2019

Abstract
In contrast to the precise monitoring of the dynamics of the leukemic clone after diagnosis, little is known about the presence, dynamics and evolution of preleukemic cells before the disease onset. We have examined this issue in 2 groups of patients: (1) firstly with MLL positive secondary leukemia (sAL), where bone marrow (BM) aspirates were available due to the follow-up of the primary disease; and (2) secondly, in cases with slow onset leukemia (SOL), that presented initially with atypical aplastic anemia (at least 30 days prior to the ALL diagnosis), and thus prediagnostic BM samples were taken and archived.
We initially selected 8 cases of SOL and 5 cases of sAL harboring both typical (MLL/AF9) and atypical (MLL/MAML2, MLL/FOXO3A, MLL/ME2) MLL fusion. In all the aforementioned patients at least one BM sample preceding the diagnosis was available. To reveal leukemospecific markers (Ig/TCR or MLL rearrangements, hyperdiploidy, ETV6/RUNX1, BTG1 deletion) and to provide better characterization of the leukemic clone the diagnostic samples were investigated using real time PCR (qPCR) as well as by SNP array and FISH. Backtracking of the patient specific clonal markers was perfomed on prediagnostic BM samples, using various methods – qPCR, FISH and NGS.
Leukemospecific markers were detected in all patients at the time of diagnosis. In the sAL cases, backtracking in the (pre)leukemic cells was successful only in patients harboring atypical MLL fusions (MAML2, FOXO3A and ME2). In contrast, in sAML patients harboring typical MLL/AF9 fusion, the MLL gene rearrangement was not detected in any of the prediagnostic samples (11 and 12 months prior to sAL onset).
In 2 sALL patients, we detected the presence of the MLL fusion in 3 or more samples before the sAL diagnosis, revealing a surprising dynamic in the size of the preleukemic clone: remarkably high initial levels (up to 90% of MLL+ cells in samples taken 15 and 17 months before the sAL diagnosis) were followed by a steep decrease in the MLL positivity levels before sAL onset. Furthermore, the MLL positivity was not restricted to lymphoid lineage, suggesting the involvement of a multipotent cell originally affected by MLL rearrangement. Similar dynamics were observed in the group of SOL cases that included patients with ETV6/RUNX1 fusion (n=2) or hyperdiploid leukemia (n=2).
In SOL cases (n=8), at least one diagnostic rearrangement was detected in preleukemic samples. The dynamics of the preleukemic cells negatively correlated with the dynamics of the non-malignant TCRG Vg9-Jg1.2 clone; raising the question of anti-tumor immunity mechanisms in the development of AL. However, this negative correlation was not proved in the group of patients with MLL positive sAL, pointing out the potential differential impact of the various leukemic aberrations in any anti-tumor immunity response.
To define additional aberrations that could help distinguish the preleukemic clone from the leukemic one, SNP array and subsequent FISH analysis were perfomed. In 4 cases where preleukemic cells were backtracked successfully, other diagnostic genetic aberrations were considered to be specific solely for leukemic cells, as they were first detected in the diagnostic samples. In the ETV6/RUNX1+ patient, 6 additional deletions were present at diagnosis (1p33, 12q13.2, 12q24.11, 12q24.32, 14q24 in 100% and 4q31 in 86% ETV6/RUNX1+cells). While the deletion of the non-affected ETV6 (12q13.2) was present in all the ETV6/RUNX1+ cells already 3 months before the diagnosis, the other diagnostic deletions comprised only 20-95% of the ETV6/RUNX1+ prediagnostic clone demonstrating significant clonal selection shortly before the leukemia onset.
In conclusion, the preleukemic cells observed in sAL and SOL cases show specific dynamics with respect to the size of the preleukemic and leukemic clone. Furthermore, we postulate that the dynamics can be modulated by immunologic mechanisms and can be dependent on genetic subtype. Moreover, typical and rare MLL fusions have different dynamics indicating that they are possibly driving different processes preceding the development of overt disease. Finally, preleukemic cells can coexist even in unexpectedly high proportions with sustained and seemingly normal hematopoiesis.
Grant support: IGA-MZ NT/12428-5, NT/14350-3, GAUK618212, RVO-VFN64165, UNCE 204012, RVO-FNM64203.

Disclosures:
No relevant conflicts of interest to declare.