Next-generation sequencing and FISH studies reveal the appearance of gene mutations and chromosomal abnormalities in hematopoietic progenitors in chronic lymphocytic leukemia

Abstract

Background: Chronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent studies have shown that the genetic alterations may appear in CD34+ hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored.

Methods: Amplicon-based deep next-generation sequencing (NGS) studies were carried out in magnetically activated-cell-sorting separated CD19+ mature B lymphocytes and CD34+ hematopoietic progenitors (n = 56) to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88, and XPO1 genes. In addition, ultra-deep NGS was performed in a subset of seven patients to determine the presence of mutations in flow-sorted CD34+CD19- early hematopoietic progenitors. Fluorescence in situ hybridization (FISH) studies were performed in the CD34+ cells from nine patients of the cohort to examine the presence of cytogenetic abnormalities.

Results: NGS studies revealed a total of 28 mutations in 24 CLL patients. Interestingly, 15 of them also showed the same mutations in their corresponding whole population of CD34+ progenitors. The majority of NOTCH1 (7/9) and XPO1 (4/4) mutations presented a similar mutational burden in both cell fractions; by contrast, mutations of TP53 (2/2), FBXW7 (2/2), and SF3B1 (3/4) showed lower mutational allele frequencies, or even none, in the CD34+ cells compared with the CD19+ population. Ultra-deep NGS confirmed the presence of FBXW7, MYD88, NOTCH1, and XPO1 mutations in the subpopulation of CD34+CD19- early hematopoietic progenitors (6/7). Furthermore, FISH studies showed the presence of 11q and 13q deletions (2/2 and 3/5, respectively) in CD34+ progenitors but the absence of IGH cytogenetic alterations (0/2) in the CD34+ cells. Combining all the results from NGS and FISH, a model of the appearance and expansion of genetic alterations in CLL was derived, suggesting that most of the genetic events appear on the hematopoietic progenitors, although these mutations could induce the beginning of tumoral cell expansion at different stage of B cell differentiation.

Conclusions: Our study showed the presence of both gene mutations and chromosomal abnormalities in early hematopoietic progenitor cells from CLL patients.

Keywords: Chromosomal abnormality; Chronic lymphocytic leukemia; FISH; Hematopoietic progenitors; Mutation; Next-generation sequencing.

Fig. 1

Cytogenetics and molecular characteristics of CD19+ and CD34+ cells. In the heatmap, rows correspond to the indicated alterations and each column represents individual CLL samples. Color-coded based on the gene and cytogenetic status (dark gray, altered; light gray, not-altered; white, not analyzed). For IGHV status: dark gray, unmutated; light gray, mutated. *Only mutations with VAF > 10% were considered for MACS isolated CD34+ cells considering the purities obtained. **Mutations in CD34+CD19− cells were assessed by ultra-deep NGS, considering mutations with VAF > 2%, taking into account the cell purities obtained from FACS sorting

Fig. 2

Mutational burden in CD19+ (dark gray) and CD34+ (light gray) cells from CLL patients. a NOTCH1 and XPO1 mutational burdens are similar in CD34+ and CD19+ cell populations. b TP53, FBXW7, and SF3B1 mutational burdens were lower in the CD34+ cell population

Fig. 3

Schematic model of events in hematopoiesis in CLL patients. Red gene names indicate the moment of appearance of mutations. Black names indicate the presence of a gene mutation in an specific cell population whereas underlined black gene names indicate the moment of expansion of tumoral cells harboring these gene mutations during B-CLL differentiation