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

Abstract

BackgroundChronic 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.MethodsAmplicon-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.ResultsNGS 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.ConclusionsOur study showed the presence of both gene mutations and chromosomal abnormalities in early hematopoietic progenitor cells from CLL patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-017-0450-y) contains supplementary material, which is available to authorized users.

Quality Assurance/Quality Control of Fluorescence in Situ Hybridization Tests in Hematologic Malignancies

“…Fluorescence in situ hybridization (FISH) is simple, reliable and cost-effective and therefore FISH is a major technology that is widely applied for clinical diagnosis, especially for hematologic malignancies, even in the era of next-generation sequencing (NGS) [1][2][3][4]. In the Clinical Cytogenetics Laboratory at MD Anderson Cancer Center, over 15,000 FISH tests are performed each year, including approximately 1,000 BCR-ABL1 and 500 MYC FISH tests, respectively.…”

Mature lymphoid malignancies: origin, stem cells, and chronicity

“…Several recent investigations have identified new pieces of this puzzle, with different conclusions drawn. [19][20][21][22][23][24] Next-generation sequencing (NGS) of sequential paired tumor samples point to ancestral clones that initiate both the first lymphoid tumor and subsequent relapse tumor. [25][26][27][28] In addition, several case stories report the onset of identical lymphoid malignancy in both donor and recipient after allogeneic hematopoietic stem cell (HSC) transplantation.…”

Age-related clonal hematopoiesis and monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia: a new association?

“…Third, a number of somatic mutations were found in both the MBL/CLL cells and PMN, supporting the idea that the MBL/CLL clone stemmed from a common ancestral hematopoietic precursor that was able to participate in both lymphoid and myeloid differentiation. By documenting that the DNA from PMN was free from contamination by MBL/CLL DNA, for example, by using molecular minimal residual disease methods relying on the individual patient's specific immunoglobulin gene rearrangements, the Authors have provided further evidence of this important finding which, although previously reported, 5,6 had remained a subject of debate. Several hematologic malignancies, including CLL, multiple myeloma (MM) and acute myeloid leukemia (AML), have well-defined precursor states that precede the development of overt cancer.…”