Hematopoietic Stem and Progenitor Cells in Patients with Mature B-Cell Malignancies Are Quantitatively and Qualitatively Deregulated Compared to Healthy Controls

Mature B-cell lymphomas represent a heteregenous group of malignancies, which are considered to arise along different steps of B-cell development, particularly from germinal or post-germinal center B-cells. Recent studies, however, indicate that hematopoietic stem and progenitor cells (HSPC) could be involved in the pathogenesis of these diseases.

We analyzed HSPC populations from 131 patients with mature B-cell malignancies, including chronic lymphocytic leukemia (CLL, n=20), mantle cell lymphoma (MCL, n=26), diffuse large B-cell lymphoma (DLBCL, n=35), follicular lymphoma (FL, n=25), and multiple myeloma (MM, n=25). For comparison, HSPC populations obtained from 22 healthy donors were used. Hematopoietic stem cells (HSC, Lin-CD34+CD38-CD90+CD45RA-), multipotent progenitors (MPP, Lin-CD34+CD38-CD90-CD45RA-), multi-lymphoid progenitors (MLP, Lin-CD34+CD38-CD90-CD45RA+), and pro-B cells (CD34+CD38+CD10+CD19+) were analyzed by flow cytometry. Proportions of HSPC and pro-B cells were related to CD34+CD38- and CD34+CD38+ cells, respectively. HSC and pro-B cell populations were sorted directly into tubes containing lysis solution for subsequent gene expression analyses. Preamplified cDNA was used for quantitative PCR of selected gene targets (n=28 in HSC, and n=27 in pro-B). The expression of targets was normalized to GAPDH expression. Corresponding cell populations isolated from healthy donors were used as controls.

The aforementioned flow cytometry analyses revealed significantly decreased MLP population in all 5 tested diagnoses: CLL (p<0.0001), MCL (p=0.0004), DLBCL (p=0.002), FL (p=0.004), and MM (p=0.004) compared to CTRL (Figure 1). Pro-B cell populations were also decreased in all tested diagnoses, but statistical significance was reached only in MCL (p<0.0001), DLBCL (p=0.0003), and MM (p=0.0025), but not in CLL (p=0.078) or FL (p=0.308). HSC populations were significantly increased in DLBCL (p=0.0023), FL (p=0.015), and MCL (p=0.036), but not in MM (p=0.207) or CLL (p=0.875). The only significant change in MPP population was an increase in CLL (p=0.031).

To gain more insight into biology of the stem and progenitor cell populations we analyzed gene expression changes of the key transcriptional regulators, HSC and pro-B cell specific surface markers, or genes that are overexpressed in particular lymphoma subtypes. We observed: 1) Significant upregulation of BCL11A, RUNX1, IKAROS, GATA2, PROM1, and CD44 mRNA within HSC populations obtained from all tested diagnoses compared to CTRL-HSC. 2) None of the targets in pro-B cells were significantly deregulated across all 5 diagnoses, the only significantly upregulated genes were IKAROS and EBF1 in CLL compared to CTRL pro-B cells. 3) NOTCH1 and CCND1 mRNA were not detected in CTRL-HSC, but NOTCH1 was detectable in more than 50% of DLBCL and MM-HSC samples, while CCND1 was detectable in more than 50% of CLL-HSC samples. 4) More than 50% difference in gene expression frequency between CTRL and patient HSC samples was observed in the case of BMI1 (CLL, DLBCL, FL and MM), FOXO1 (MM), FOXP1 (DLBCL), and IRF4 (CLL). 5) Pro-B cells obtained from CTRL samples did not express BCL2, BMI1, MYC, PAX5, or ZAP70, but these genes were detected in more than 50% of patient pro-B cell samples as follows: BCL2 in FL, BMI1 and ZAP70 in CLL and DLBCL, MYC in CLL, DLBCL and FL, PAX5 in CLL. 6) More than 50% difference in gene expression frequency between CTRL and patient pro-B cells was observed in case of BCL11A (CLL, DLBCL, FL, MM, and MCL), BCL2L1 (CLL, DLBCL, FL, MM), CD38 (CLL, DLBCL, FL), CD44 (in CLL, DLBCL), IRF4 (CLL, DLBCL, FL), IRF8 and LEF1 (CLL), PU.1 (CLL, FL), and RUNX1 (CLL, DLBCL).

In this study, we showed that bone marrow HSPC in patients with mature B-cell malignancies are quantitatively and qualitatively different compared to HSPC obtained from healthy controls. We currently study whether this deregulation is caused by the influence of malignant cells on HSPC or reflects HSPC intrinsic changes that are involved in the pathogenesis of these malignancies.

Grant support: IGA-MZ NT13201-4/2012, GACR14-19590S, UNCE 204021, SVV-2013-266509, GA-UK 595912, PRVOUK-27/LF1/1

Figure 1:

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Percentage of HSPC in patients with diverse mature B-cell malignancies and healthy donors (controls). Symbols are plotted at means and standard deviations are omitted for clarity. Asterisk denotes statistically significant changes.

Figure 1:

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Percentage of HSPC in patients with diverse mature B-cell malignancies and healthy donors (controls). Symbols are plotted at means and standard deviations are omitted for clarity. Asterisk denotes statistically significant changes.

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