Next-Generation Sequencing Reveals Oncogenic Driver Mutations In Both Progressive and Non-Progressive Monoclonal B Lymphocytosis

Essentially all cases of CLL are preceded by an asymptomatic expansion of a monoclonal B cell population, a condition that has been termed monoclonal B-cell lymphocytosis (MBL). MBL can be found in the peripheral blood of 3 to 5% of normal older adults. Although MBL is precursor state of CLL, the majority of MBL do not progress to CLL. Thus, MBL is being increasingly recognized and diagnosed in routine clinical practice, but the prognostic and treatment implications of MBL are unclear.

The application of whole genome and exome sequencing has yielded important insights into somatic genetic mutations that contribute to the biology of CLL. Such work has identified a role for recurrent mutations in a number of novel genes including NOTCH1 and SF3B1 in CLL. However, the role of genetic mutations in the development of MBL and progression of MBL to CLL is completely unknown. Our central hypothesis is that acquired somatic mutations are responsible for the progression of MBL to CLL and determine an individual patient’s disease course. Therefore, we applied exome sequencing to a cohort of MBL patients to understand the mutation profile of pre-emergent CLL and the determinants of progression from MBL to CLL.

Thirty-six clinical MBL (CD19⁺ cell count: 0.5 – 5.0 x10⁹/L) cases were ascertained through flow cytometric screening of a population based cohort of non-hematology patients identified via an absolute lymphocytosis on a routine complete blood count. Thirteen cases had a follow-up sample obtained at an average time of 4.8 years after initial study enrollment. All cases had a detectable CLL-phenotype clone but did not meet iwCLL criteria for a diagnosis of CLL. Samples with clinical follow-up were characterized as progressive or non-progressive based on whether the CLL phenotype clone expanded at the time of follow-up. CLL-phenotype cells and non-malignant monocytes were purified using FACS and DNA was extracted. We performed whole-exome sequencing using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13).

A total of 86 exomes were sequenced for this study. Seven MBL cases were progressive and 29 MBL cases were non-progressive. Paired normal DNA was sequenced for all of these cases. Variants were filtered both through pair-wise comparisons of MBL and monocyte sequencing data and by prioritizing acquired mutations identified in know tumor suppressor or oncogenes. In all, we generated over 8 GB of sequencing data using high throughput sequencing on the Illumina platform. The number of mutations per exome were similar between progressive and non-progressive cases. Numerous non-synonymous variants in known tumor suppressor or oncogenes were identified in non-progressive cases including: NOTCH2, PIK3R1, SYK, MLL2, CDK12, CIITA, and CD79A. Non-synonymous variants identified exclusively in progressive cases included CREBBP and BRCA2.

This study represents the first in-depth investigation of MBL exomes. Our data show that individual events in known oncogenes and tumor suppressor genes occur commonly in non-progressive MBL. Their presence does not indicate poor prognosis. These findings imply that sequential oncogenic mutations and combinations of specific genetic events are likely required for the progression of MBL to CLL. The knowledge of genetic variants that collectively contribute to the development of CLL would allow the development of risk stratification strategies for patients with MBL.