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

Chronic lymphocytic leukemia (CLL) is characterized by the clonal proliferation and accumulation of neoplastic B lymphocytes in the blood, bone marrow, lymph nodes, and spleen [ 1, 2]. Immunophenotype analysis of CLL cells shows expression of CD5 T cell antigen as well as CD19, CD20, and CD23 B cell surface antigens [ 3]. In molecular terms, CLL is defined by the presence of chromosomal abnormalities (11q-, +12, 13q-, 17p-) that play an important role in CLL prognosis [ 4]. The mutational status of the immunoglobulin heavy chain ( IGHV) is also considered a prognostic marker in CLL [ 5, 6]. Recently, the development of next-generation sequencing (NGS) techniques has enabled mutations to be identified in novel target genes in CLL [ 7, 8], and mutations in some drivers such as NOTCH1, SF3B1, TP53, and MYD88 genes have been shown to have a prognostic impact in CLL patients [ 9– 11].

The cellular origin of this disease remains controversial [ 12– 14]. Recent studies have reported that CLL pathogenesis may start at a previous maturational cell stage, or even in hematopoietic stem cells (HSCs). Fluorescence in situ hybridization (FISH) studies showed that +12 and 13q- abnormalities are present in CD34+CD19− cells, suggesting that these common chromosomal abnormalities could appear in HSCs [ 15, 16]. Interestingly, xenotransplantation studies reported that HSCs from CLL patients were able to reproduce the CLL phenotype in murine models [ 17]. In addition, CLL mutations may appear in HSCs, supporting the idea that CLL pathogenic events occur at an early stage of the hematopoietic process [ 18].

Taking the previous studies in this field into account, it is well known that chromosomal abnormalities as well as gene mutations are important events in CLL pathogenesis [ 19]. However, it is still not clear which genetic events are related with the origin of the disease and when these alterations occur and have a functional impact inducing tumoral cell expansion during B cell differentiation. For these reasons, in this study, chromosomal abnormalities and gene mutations in hematopoietic progenitors were analyzed, showing that the whole population of CD34+ progenitors, even at the level of CD34+CD19−, are already affected at genetic level in CLL patients. In particular, mutations of FBXW7, MYD88, NOTCH1, and XPO1 as well as 11q and 13q deletions were detected in CD34+ progenitors. By contrast, the origin of TP53 and SF3B1 mutations and IGH alterations could take place at a later maturational stage. Apart from B lymphocytes, some of these genetic alterations were also observed in other mature cell fractions (T lymphocytes and monocytes) derived from HSCs. Integrating all these results, a pattern of appearance and expansion of these genetic events during B-CLL cell differentiation was suggested.

Our data provide evidence of the presence of mutations and chromosomal abnormalities in early hematopoietic progenitors in BM samples of CLL patients. These results shed light on the cell of origin of CLL in a previous developmental stage to mature B cells, demonstrating CLL patients can also show genetic events in the CD34+ hematopoietic progenitors. These results are consistent with the findings of two recent studies [ 17, 18]. MACS isolation was performed on the whole population of CD34+ cells including CD34+CD19+ pro-B cells. A previous study has reported that the pro-B cell population is larger in the bone marrow of CLL patients than in healthy donors (mean range of 18% pro-B cells in the total bone marrow CD34+ cell count, exceeding 30% in some cases) [ 17]. This suggests that the patients who presented a mutational burden of < 30% in the CD34+ cell population may only harbor the mutation in CD34+CD19+ pro-B cells, rather than in the early hematopoietic progenitors. It is of particular note that 10 out of 15 patients with mutations in the whole population of CD34+ cells had mutational rates of > 30%, suggesting that these mutations not only appear in the pro-B cells, but also at earlier maturational stages of B cell differentiation (CD34+CD19− progenitors). In order to assess this hypothesis, we could perform ultra-deep NGS studies of flow-sorted CD34+CD19− cells using PB samples in a small subset of patients within the main cohort, detecting that all the mutations, except to one, were already present in this cellular fraction. Therefore, these results confirmed that mutations on CLL driver genes could occur in early hematopoietic progenitor cells of these patients. In particular, our sequencing results suggest that mutations in NOTCH1, MYD88, FBXW7, and XPO1 may appear in CD34+CD19− cells whereas TP53 and SF3B1 mutations could appear in a later stage of B cell differentiation. As far as we are concerned, these results were demonstrated for the first time in fresh hematopoietic progenitor cells without having been cultured.

As these driver mutations have been detected in early hematopoietic progenitors of some CLL patients, we hypothesized that these alterations can affect hematopoietic lineages other than B cells. As it was previously reported, some mutations in well-known CLL drivers can also appear on a low percentage in other mature cell fractions as CD3+ T lymphocytes and/or CD14+ monocytes [ 18].

In order to determine the stage of B cell differentiation in which these mutations induced an expansion of the tumoral cell population, first, the mutational burdens in CD19+ mature B lymphocytes and CD34+ progenitors were compared. Since the mutational burden in CD34+ cells with NOTCH1 mutations was as high as that observed in CD19+ cells in most of the cases, it could be hypothesized that mutations in this gene induced an expansion of the CLL hematopoietic progenitors. Indeed, ultra-deep NGS studies confirmed this in patient ID-42 who had NOTCH1 mutation in more than 10% of CD34+CD19− cells. Moreover, every XPO1 mutation observed in our cohort was present in the CD34+ progenitors and the mutational burden remained similar in both cellular fractions. However, the percentage of XPO1 mutations in the CD34+ cells exceeded 30% in very few cases, suggesting that these mutations could be enriched at an intermediate B cell stage as CD34+CD19+ pro-B cells. On the other hand, the mutational burdens of TP53, SF3B1, and FBXW7 were considerably lower in the CD34+ cell population. Although SF3B1 mutation was not detected in CD34+CD19− cells, suggesting it as a late event in B-CLL differentiation, one out of four SF3B1 mutated patients (ID-43) carried a mutation in a high percentage of the whole population of CD34+ progenitors, similar to results from a previous study [ 18]. Besides this, alterations in this gene have been also reported in CD34+ cells of patients with myeloid malignancies [ 33]. Therefore, it should be essential to study larger cohorts of CLL patients in order to determine what type of SF3B1 mutations occur in HSCs of CLL patients and functional studies to assess the differences between “CLL-HSCs SF3B1 mutated” and “MDS-HSCs SF3B1 mutated.”

Focusing on the presence of cytogenetic abnormalities in CD34+ cell populations in our cohort, 11q- and 13q- appeared in the CD34+ progenitors at high percentages, as reported previously [ 15, 16], supporting the hypothesis that these chromosomal aberrations could be an early event in CLL [ 8]. By contrast, IGH alterations were not present in any of the CD34+ hematopoietic progenitors. Previous case report studies have yielded similar results [ 34, 35], suggesting that IGH alterations occur in an advanced stage of the lymphocyte maturation process.

The analysis of patients with more than one genetic alteration allowed us to define a hierarchy of the appearance of these genetic events. When IGH alterations and the NOTCH1 mutation are present in the same patient (ID-02), it is clear that the NOTCH1 mutation is an earlier step than the IGH alteration during B cell differentiation. Moreover, when 11q-, 13q-, and XPO1 mutations are present in the same patient (ID-21), they all appear in the CD34+ progenitors. However, 11q- and 13q- are certainly present at a higher allele frequency than the mutational load of XPO1, suggesting that cells with 11q- and 13q- were expanded in an earlier stage than the XPO1 mutation in the pathogenesis of the disease [ 8]. Specifically, the dominant clone in the two cases with a double mutation in NOTCH1 and XPO1 (ID-34 and ID-49) was always NOTCH1 rather than XPO1. Therefore, since XPO1 is still present in a small part of the CD34+CD19− cell population and greatly enriched on the total CD34+ fraction, we may consider that cells carrying XPO1 mutations are expanded in an intermediate event of B-CLL differentiation, given the previous possible events such as 13q- and 11q- abnormalities or MYD88 and NOTCH1 mutations. Taking into account all these results, a model of the appearance of genetic events during the hematopoiesis in CLL has been suggested (Fig.  3). However, as some of these genetic alterations were observed in few cases, it would be interesting to sequence all cell fractions in larger cohorts of patients.

The prognostic impact of gene mutations and chromosomal abnormalities in CLL has been well characterized in several studies of large cohorts of patients [ 4, 9, 23, 36]. TP53, NOTCH1, and SF3B1 are described as poor prognostic mutations [ 10, 37– 40]. The prognostic impact of the presence of mutations in CD34+ cells from CLL patients has been assessed for the first time in this study, showing that CLL patients harboring mutations in the hematopoietic progenitors showed worse prognosis. It is essential to achieve a better understanding of these results since only six genes were analyzed in our study, whereas a CLL exome exhibits an average of 20 mutations [ 8, 10]. Therefore, further studies analyzing the whole exome in larger cohorts should be performed in order to accurately define their impact. In addition, conventional CLL therapies may not be able to eradicate or reduce the malignant CD34+ CLL clone and may be responsible for the relapse of patients bearing mutations in these cells. Given that these therapies seem not to have a clear effect on CD34+ CLL cells, allogeneic stem cell transplantation could be an option for overcoming the challenges that may arise from CD34+ cell treatment [ 41].

Our data show that recurrent CLL chromosomal abnormalities and gene mutations are present not only in mature B lymphocytes but also in hematopoietic progenitors. Although CLL is a clonal mature B cell disease, our results provide strong evidence that CLL may originate in the early stages of hematopoiesis. Both chromosomal alterations and point mutations are highly relevant to the disease pathogenesis, with a clinical impact as soon as they appear. To the best of our knowledge, our study is the first to analyze both genetic events in different cellular fractions of the same patients by NGS and FISH. It provides us with a broader understanding of CLL initiation and development, opening up possibilities for future therapies.