Clones with and without Sensitivity Towards Treatment In Vivo Co-Exist within the Tumor Cells of a Single Patient with ALL
Authors: Finkenzeller Cornelia , Carlet Michela , Vosberg Sebastian , Ziegenhain Christoph , Neumann Martin , Riecken Kristoffer , Cornils Kerstin , Fehse Boris , Baldus Claudia D. , Enard Wolfgang , Greif Philipp A. , Jeremias Irmela 
Published: October 2019

Abstract
Acute lymphoblastic leukemia (ALL) is known to consist of several clones that might have different chromosomal, genetic or epigenetic aberrations. However, little is known about functional diversity in these different clones. In some patients, cells cannot be eradicated by standard therapy regimens, and aggressive or otherwise unfavorable clones might survive, eventually resulting in relapse and a poor prognosis of the patients.
Here, we asked whether genetically distinct clones of ALL from a single patient would show a functionally distinct response towards drug treatment in vivo.
As technical approach, we genetically engineered primary patients' ALL cells growing in immuno-compromized NSG mice as patient derived xenograft (PDX) cells by lentiviral transduction. ALL PDX cells were red-green-blue (RGB) color marked in order to discriminate several differently colored cell populations in the same mouse in functional in vivo experiments. ALL PDX cells further expressed luciferase for bioluminescence in vivo imaging (BLI) for sensitive and reliable monitoring of disease burden. Limiting dilution transplantation of RGB marked PDX cells transplanted into groups of mice allowed generating individually marked single cell clones which were discriminated by flow cytometry. Populations expressing a distinct color were sorted and analyzed by ligation mediated PCR to verify distinct integration of lentiviral inserts to prove single cell clone (SCC) origin of the population. In sum, eight distinct SCCs could be generated and were used for functional and -OMICs approaches.
Targeted resequencing of the eight SCCs and the bulk cells revealed that all samples had mutations in CSMD1 and HERC1 with variant allele frequencies (VAF) of 0.5, indicating that these mutations represent the founding clone. However, we also found mutations that were only present in single samples: FAT1 and STAG2 mutations were found in SCC 3, whereas CSMD1 and USP6 mutations were found in SCC 6. Whole exome sequencing revealed SCC specific patterns, identifying SCC 6 being the clone furthest away from the bulk population.
As the patient showed a high hyperdiploidy (+6,+13,+14,+17,+18,+21,+22,+X), we tested SCC and bulk cells by fluorescence in situ hybridization (FISH) and found that both the bulk sample and the SCCs consisted mainly of cells harboring three X chromosomes and to a minor proportion (between 2% and 20%) of cells harboring two X chromosomes. Only SCC 6 consisted exclusively of cells harboring two X chromosomes. Additionally, this SCC showed a distinct DNA-methylation pattern analyzed by 450K arrays (illumina).
To analyze if the chromosomal, genetic and epigenetic differences also resulted in functional diversity, we first performed a competitive transplantation assay, injecting a mixture of five SCCs in the same ratio (20% each) into single mice. After 42 days when overt leukemia had established in the mice, cells were re-isolated and proportion of SCCs reanalyzed according to their specific color. Interestingly, SCC 5 (25%) and 7 (36%) had a clear growth advantage over SCCs 1 (14%), 6 (13%) and 8 (12%). The same pattern could be overserved if only SCC 5 (50% in, 92% out) and SCC 6 (50% in, 8% out) were transplanted.
Next, response towards chemotherapeutic drugs was assessed. In vitro, SCC 6 was much more resistant towards the glucocorticoids prednisolon and dexamethasone (Dexa) compared to all other SCCs and bulk cells. Cells of SCC 5 and SCC 6 were mixed in equal amounts and transplanted into mice. Four days after transplantation, mice were randomized and treated with PBS or Dexa (2 or 8 mg/kg i.p., 5 days a week, 5 weeks). BLI showed a clear response towards therapy of the entire tumor. After 61 days, control treated mice showed again an outgrowth of SCC 5 (83% vs. 17% SCC 6), while Dexa treated animals showed the opposite pattern (Dexa 2 mg/kg: SCC 6 35%; Dexa 8 mg/kg: SCC 6 59%) indicating that SCC 6 was more resistant towards Dexa treatment in vivo.
Taken together, our results clearly show that within a single ALL patient, genetically and functionally distinct subpopulations exist. Combining PDX model with genetic marking of the cells enables us to in-depth analyze SCCs of a single patient sample and eventually identify adverse prognostic markers.

Disclosures
No relevant conflicts of interest to declare.