Preneoplastic somatic mutations including MYD88L265P in lymphoplasmacytic lymphoma

Abstract

Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated MYD88. We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found MYD88^L265P in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated MYD88 in B cell precursors and BCL2 overexpression. Thus, MYD88^L265P is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas.

Fig. 1.. Mutations during B cell lymphopoiesis in elderly individuals and patients with LPL/WM.

(A) Schematic of workflow, including subject disposition, BM cell types isolated by FACS, and methodology used for WES. (B) Average numbers and range of somatic mutations in CD34-positive HPCs, B cell precursors, and mature B lymphocytes from elderly individuals (N = 5) and patients with LPL/WM (N = 10). Colors indicate types of mutations. (C) Absolute numbers and percentages of shared mutations across the different stages of B cell lymphopoiesis in elderly individuals and patients with LPL/WM. (D) Rainfall plot of somatic mutations detected in cells from the different stages of B cell lymphopoiesis from elderly individuals and patients with LPL/WM. Mutations are ordered from left to right along the x axis from the first variant on the short arm of chromosome 1 (chr1) to the last variant on the long arm of chromosome Y. The vertical axis shows the distance between each mutation and the previous one on a logarithmic scale. Density of mutations, based on the number and intermutation distances, is shown across the top of each rainfall plot.

Fig. 2.. MYD88^L265P in normal B cell precursors and B lymphocytes from patients with LPL/WM.

(A) Frequency of MYD88^L265P mutation, as assessed by WES and by ASO-PCR or droplet digital PCR (dd-PCR), in CD34-positive HPCs, B cell precursors, mature B lymphocytes, tumor B cells, and PCs from patients with LPL/WM. (B) Presence of wild-type (WT) versus heterozygous (HET) or homozygous (HOM) MYD88^L265P in 10,891 CD34-positive HPCs, B cell precursors, and mature B lymphocytes isolated by FACS from four patients with LPL/WM and stained with oligo-coupled mAbs targeting CD10, CD19, CD20, CD27, CD34, and CD38. Color scale indicates the intensity of antigen expression.

Fig. 3.. Genetic evolution during lymphomagenesis: Shared and private somatic mutations in normal cells and tumor cells from patients with LPL/WM (n = 10).

(A) Schematic representation of genetic evolution from CD34-positive HPCs, B cell precursors, and mature B lymphocytes to tumor B cells and PCs. Somatic mutations and CNAs that were private to, or shared between tumor B cells and PCs, are shown in the Venn diagrams. (B) UpSet plot summarizing the distribution of somatic mutations present in normal and tumor cells from 10 patients with LPL/WM. Black dots and vertical connecting black lines indicate the sharing of somatic mutations by two or more cell types, represented by the dots. Individual dots represent somatic mutations that are private to the respective cell type. Average numbers of somatic mutations shared by the different combinations of cell types or private to a specific cell type are shown in the top, ordered from lowest to highest number. Bars representing average numbers of somatic mutations shared by two, three, four, or all five cell types are colored red, green, blue, and light blue, respectively. The average numbers of somatic mutations per cell type are indicated on the right side of the plot.

Fig. 4.. Intratumor heterogeneity in, and malignant transformation to, LPL/WM.

(A) Schematic of workflow, including subject disposition, BM cell types isolated by FACS, and methodology used for simultaneous scRNA/BCR-seq. (B) Uniform manifold approximation and projection (UMAP) of 42,204 B cells and PCs isolated by FACS from three individuals with IgM MGUS and three patients with LPL/WM. Cells with predominant (clonal) BCR gene rearrangements are represented by specific colors. (C) UMAP of cells carrying the clonal Ig gene sequence. B cell precursors, mature B lymphocytes, and PCs are represented by specific colors. (D) UMAP of nonclonotypic cells. B cell precursors, mature B lymphocytes, and PCs are represented by specific colors. (E) Distribution of cells according to maturation stage (i.e., B cell precursors, mature B lymphocytes, and PCs) based on scRNA/BCR-seq (considering clonotypic plus nonclonotypic cells and only clonotypic cells) and multidimensional flow cytometry (MFC). (F) Volcano plot displaying differentially expressed genes found in clonotypic cells from individuals with IgM MGUS and patients with LPL/WM. Each dot corresponds to an individual gene. Differentially expressed genes with a log₂ fold change of ≤0.2 and P_adj < 0.0005 are colored red. MS4A1 (CD20) is highlighted because of its ascribed role in defining early versus late stages of B cell differentiation. BCL2, CXCR4, and HIF1A are highlighted because of their ascribed role in the pathogenesis of LPL/WM.

Fig. 5.. Mutated MYD88 alone is insufficient to induce lymphoma in mice.

(A) Schematic representation of the transgenic mouse models used in these experiments. (B) Relative numbers (versus controls) of B cells, plasmablasts (PBs), and PCs in the spleen and BM of mice euthanized at 5, 10, and 15 months of age (n = 6 mice per cohort); *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Kaplan-Meier distributions of overall survival (OS) for the three transgenic mouse models and control mice, plus median OS (mOS) for each. (D) Frequencies of tumor locations in the three transgenic mouse models. (E) Flow cytometry analyses showing the percentages of B cells, plasmablasts, and PCs in the BM, lymph nodes, and salivary glands of control mice and the M88Ym and M88Yc mouse models (n = 6 mice per cohort); *P < 0.05 and ***P < 0.001. Data were obtained from M88Ym and M88Yc mice euthanized upon symptoms or signs of disease (between 18 and 24 months) and were compared with age-matched Ym and Yc mouse controls. (F) Representative histopathologic staining with hematoxylin and eosin (H&E) and B220 and CD138 mAbs in lymph nodes from M88Ym mice and in salivary glands from M88Yc mice.

Fig. 6.. BCL2 and BCR signaling cooperate with MYD88 in LPL/WM development in mice.

(A) Schematic representation of M88B2m and M88BCRm transgenic mice. (B) Kaplan-Meier distributions of OS for M88B2m, M88BCRm, M88Ym, and control mice, plus median OS for each. (C) Frequencies of tumor locations in M88B2m and M88BCRm transgenic mice. (D) Flow cytometry analyses showing the percentages of B cells, plasmablasts, and PCs in the BM, lymph nodes, and salivary glands of M88B2m and M88BCRm mice versus control mice (n = 6 mice per cohort); **P < 0.01 and ***P < 0.001. (E) Representative histopathologic staining with hematoxylin and eosin and B220 and CD138 mAbs in the BM of M88B2m mice and in lymph nodes of M88BCRm mice. (F) Serum electrophoresis and IgM levels by enzyme-linked immunosorbent assay in M88B2m and M88BCRm mice compared to M88Ym, M88Yc, and control mice; *P < 0.05 and **P < 0.01. (G and H) Median number of CNAs (G) and somatic mutations (H) per tumor in mice with single MYD88 mutation (M88Ym and M88Yc) versus those with MYD88 mutation and secondary genetic alterations (M88B2m and M88BCRm); *P < 0.05.

Fig. 7.. Monitoring treatment efficacy based on the presence of MYD88^L265P.

(A) Mature B lymphocytes (n = 2497) from one patient with LPL/WM, showing wild-type MYD88 and CNAs in selected regulatory regions of 6q, per scDNA-seq. (B and C) Kaplan-Meier distributions of time to progression (TTP) from time of response assessment, according to presence versus absence on ASO-PCR of MYD88^L265P in whole BM samples after treatment (B) and presence versus absence of ≥0.1% phenotypically aberrant B cells on MFC (C). (D) Number and proportion of patients with LPL/WM (N = 46) who were positive or negative for MYD88^L265P on ASO-PCR and did or did not have ≥0.1% phenotypically aberrant B cells on MFC.