Somatic MDC1 Mutation in Putative Pre-Leukaemic Stem Cell of a Biclonal Case of Chronic Lymphocytic Leukaemia
Authors: Hotinski Anya K , Lower Karen M , Kuss Bryone J. 
Published: October 2019

Abstract
Chronic lymphocytic leukaemia (CLL) is an inherently heterogeneous disease in which founding lesions and cell of origin remain a subject of debate. This case of truly biclonal CLL provides evidence of the presence of a pre-leukaemic stem cell, supporting other lines of evidence that pathogenic genetic and epigenetic lesions in CLL arise early in haematopoiesis.
An individual with trisomy 12 containing CLL, present at a frequency of 30%, was identified. Two distinct CD5/19-positive CLL clones were separated by flow cytometry, based on bimodal expression of CD49d. Each leukaemic clone was subjected to targeted immunoglobulin heavy chain variable region gene (IGHV) sequencing, whole exome sequencing (WES) and single-nucleotide polymorphism (SNP) microarray. CD5-positive T cells and a non-malignant superficial skin biopsy sample from the same individual were analysed as comparators.
The CD49d-positive and CD49d-negative fractions harboured completely different IGHV rearrangements and mutational statuses (V4-34*02, hypermutated, and V3-21*02, unmutated, respectively) indicative of two unique leukaemic clones. Furthermore, trisomy 12, a relatively common aneuploidy in CLL, was present at high frequency (89.9%) in the CD49d-positive fraction but was absent in the CD49-negative and T-cell fractions. A mutation in the known CLL driver SF3B1 (c.1866G>T) was present in the CD49-negative, unmutated clone alone, along with a 17p deleted subclone in around 20% of this fraction. The presence of a 17p deletion is highly clinically relevant in this disease, and it is noteworthy that this subclone was not detected by routine fluorescence in situ hybridisation (FISH) at diagnosis. Two putative driver mutations in KMT2D and BCL11B were detected in the CD49d-positive, trisomy 12 clone alone (c.15256C>T and c.1387G>A respectively). A TET2 mutation (c.3609C>G) was found in all three fractions. The mutation in this epigenetic regulator has previously been described in clonal haematopoiesis of indeterminate potential (CHIP) and was hypothesised to have been acquired in a haematopoietic stem cell predisposing the individual to the development of haematological malignancy. However, the presence of this mutation was confirmed by Sanger sequencing of genomic DNA extracted from the skin sample, and is therefore of germline origin. The role of germline TET2 variants has not been described in lymphoid malignancy.
Importantly, eight coding mutations were found common to both leukaemic clones but were absent from T-cells, implicating a common cell of origin prior to IGHV rearrangement but following B-lineage commitment (that is, a putative pre-leukaemic stem cell). One of these mutations was a disruptive 123 base pair deletion (c.4824_4946del) in MDC1 (mediator of DNA damage checkpoint 1) and was present at a variant allele frequency (VAF) of approximately 50% in both CLL clones, suggesting it was a founder mutation prior to subclone divergence. MDC1 is a mediator in the Ataxia Telangiectasia Mutated (ATM)-dependent DNA damage repair pathway and has not been previously implicated in CLL pathogenesis unlike ATM, which is frequently lost in del11q CLL. The proposed evolution of CLL in this case is presented in Figure 1.
In summary, this highly unusual case of CLL provides insights into the evolution of CLL and adds another line of evidence to support the proposed early origins of CLL in haematopoiesis. The role that MDC1 may contribute to CLL pathogenesis, in particular its association with ATM dysregulation, remains to be elucidated. Further work to delineate critical pathways based on the discovered mutations is underway, and includes global transciptomic analysis of the purified leukaemic clones by RNAseq.

Disclosures
No relevant conflicts of interest to declare.