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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Journal of Hematology & Oncology 1/2017

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

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2017
Autoren:
Miguel Quijada-Álamo, María Hernández-Sánchez, Cristina Robledo, Jesús-María Hernández-Sánchez, Rocío Benito, Adrián Montaño, Ana E. Rodríguez-Vicente, Dalia Quwaider, Ana-África Martín, María García-Álvarez, María Jesús Vidal-Manceñido, Gonzalo Ferrer-Garrido, María-Pilar Delgado-Beltrán, Josefina Galende, Juan-Nicolás Rodríguez, Guillermo Martín-Núñez, José-María Alonso, Alfonso García de Coca, José A. Queizán, Magdalena Sierra, Carlos Aguilar, Alexander Kohlmann, José-Ángel Hernández, Marcos González, Jesús-María Hernández-Rivas
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s13045-017-0450-y) contains supplementary material, which is available to authorized users.

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.

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Zusatzmaterial
Additional file 1: Table S1. Clinico-biological characteristics from CLL patients. Table S2. Illumina Primer Design. Table S3. Validation of mutations detected at low frequency by ultra-deep NGS in flow-sorted cell fractions using 454 sequencing. Table S4. Patients’ characteristics regarding the presence of mutations in CD34+ progenitors. Table S5. Patients’ characteristics regarding the mutational burden maintenance or decrease in CD34+ progenitors. Table S6. Treatments prior bone marrow extraction of 4 CLL patients who relapsed and correlation with the CD19+ and CD34+ mutational status. Figure S1. Representation of the purity analysis of FACS sorted cell populations. Figure S2. Kaplan-Meier analysis of overall survival (A) and time to first therapy (B) in patients with mutations in their CD34+ progenitors. (DOCX 200 kb)
13045_2017_450_MOESM1_ESM.docx
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