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A. Agathangelidis et al.
required for a clinical diagnosis of CLL (≥5x109/L).21-24 MBL, found in otherwise healthy individuals, is divided into 2 subtypes based on the number of circulating cells: ‘high-count MBL’ (HC-MBL: 0.5-5x109/L) that evolves into CLL requiring therapy at a rate of 1%/year,25 and ‘low- count MBL’ (LC-MBL: <0.5x109/L) that has not been observed to progress into a clinical disease,26 yet persists over time.26,27 Several typical CLL driver gene mutations have been reported in HC-MBL9,28,29 even years before the transition to CLL,30 and these correlate with adverse dis- ease course.31 Such mutations have been reported in mul- tipotent hematopoietic progenitor CD34+ cells from patients with CLL,32 suggesting that such aberrations may also be implicated in CLL onset.
Here, we aimed to gain insight into the genetic lesions that may be involved in the transformation from MBL to CLL, analyzing LC-MBL cases for the first time. To this end, we used whole-genome sequencing (WGS) and tar- geted re-sequencing to profile LC-MBL, HC-MBL and a particularly indolent subset of CLL, i.e. patients with ultra- stable disease for more than ten years, thus, clinically anal- ogous to MBL. Moreover, in order to explore the possible origin of genetic lesions at the hematopoietic progenitor cell level, we analyzed polymorphonuclear (PMN) cells from the study participants.
We report that the genomic profiles of ultra-stable CLL patients are very similar to individuals with LC-MBL and HC-MBL, characterized by infrequent CLL driver gene mutations that, however, were not associated with dis- ease progression. Furthermore, we observed non-coding variants (NCVs) that target key pathways/cellular process- es relevant to normal and neoplastic B-cell development, thus, potentially contributing to the leukemic transforma- tion. We also found shared somatic mutations between MBL/CLL and PMN cells, strengthening the notion that at least a proportion of somatic mutations may occur before the onset of CLL.
Methods
The research protocol was approved by the Institutional Ethics Committee and all participants gave written informed consent in accordance with the Declaration of Helsinki.
Study population
The study cohort comprised 9 subjects with LC-MBL, 13 sub- jects with HC-MBL, and 7 patients with Rai stage 0 CLL, herein called ‘ultra-stable’ CLL. Detailed information about the study cohort is provided in the Online Supplementary Appendix.
Cell samples
Chronic lymphocytic leukemia cells were stained with anti- CD19, anti-CD5 and anti-CD20 antibodies. CD19+CD5+CD20dim cells were sorted using a High Speed FACS Sorter MoFLo (Beckman Coulter) according to previously published methods.26 PMN cells were sorted based on physical parameters. Buccal cells were collected with the use of appropriate buccal swabs (Epicentre, Madison, USA).
DNA extraction
The NucleoSpin® Tissue XS kit (Macherey-Nagel, Germany) was used for DNA extraction in samples with less than 5x104 cells and the QIAamp DNA Micro kit (Qiagen, Germany) in samples with cell numbers ranging between 5x104 and 1x106. The QIAamp
DNA Blood Mini kit (Qiagen, Germany) was used for samples with more than 1x106 cells as well as for the buccal samples. DNA quantity and quality were assayed using the Qubit dsDNA HS Assay Kit (Life Technologies, USA).
WGS: library preparation
The Nextera technology was utilized for the library construc- tion (NexteraTM DNA Sample Prep Kit, Illumina, USA) as it requires low input material whilst maintaining library complexity. Fifty ng of genomic DNA were used for the construction of libraries that were sequenced in paired-end mode 2x100bp on a HiSeq 2000 (Illumina, USA).
A variant allele frequency (VAF) of 10% was used as threshold for variant calling. More detailed information regarding the bioin- formatics analysis is given in the Online Supplementary Appendix.
Targeted re-sequencing: library preparation
Probes targeting all coding exons or hotspot regions of 11 known or postulated CLL driver genes (ATM, BIRC3, MYD88, NOTCH1, SF3B1, TP53, EGR2, POT1, NFKBIE, XPO1, FBXW7) (Online Supplementary Table S1) were designed using Agilent’s SureDesign service (https://earray.chem.agilent.com/suredesign/home. htm). The target regions were captured using the HaloPlex HS tar- geting enrichment kit (Agilent Technologies, USA). Paired-end sequencing (150 bp reads) was performed on the NextSeq instru- ment with the use of the 500/550 High Output Kit (Illumina, USA).
Gene enrichment analysis
The identification of genes/gene pathways (gene enrichment analysis, GEA) enriched within the targets of NCVs and motif- breaking events caused by NCVs was performed with Enrichr31 using the KEGG 2016 gene database.
Results
WGS reveals highly similar mutational profiles in MBL and ultra-stable CLL
Whole-genome sequencing was performed on 6 individ- uals with LC-MBL, 5 individuals with HC-MBL, and 5 patients with ultra-stable CLL. For each individual/patient, samples from MBL/CLL cells and PMN cells were evaluat- ed against buccal (control) cells resulting in a total of 48 samples sequenced with an average autosomal coverage of 32X (Online Supplementary Table S2). Basic demographic and biological characteristics of the MBL/CLL cases included in the WGS analysis are provided in Online Supplementary Table S3. Overall, 37,033 somatic variants were detected in MBL/CLL samples with an average of 2040 somatic variants in LC-MBL (range: 298-2871), 2558 in HC-MBL (range: 1428-3483), and 2400 in CLL (range: 1650-3176), respectively. Notably, 2792 variants were identified in the 15 PMN control samples compared with buccal DNA, with an average of 186 variants/sample (the PMN sample from case CLL_3 was excluded from the analysis due to tumor cell contamination) (Figure 1A). Highly analogous mutation rates were observed in HC- MBL and CLL (0.79 and 0.74 mutations per Mb, respec- tively), while a slightly lower rate was seen in LC-MBL (0.63 mutations per Mb); this latter finding was due to sample LC-MBL_1 (excluding this sample, the average mutation rate for LC-MBL would have been 0.74 muta- tions per Mb) (Figure 1B). The ratio of single-nucleotide variants (SNVs)/small indels was again almost identical in
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