Clonal evolution of B cells in transformation from low- to high-grade lymphoma

Abstract

An outcome of low-grade B cell non-Hodgkins's lymphomas is the transformation to high-grade diffuse large B cell lymphomas (DLBL). To investigate the mechanisms of clonal evolution in the transformation to DLBL, we performed longitudinal molecular analyses of immunoglobulin (Ig), V(H)DJ(H) gene sequences expressed in cases of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), and follicular lymphoma (FL) that transformed to DLBL. Among the neoplastic CLL and SLL cells and their respective high-grade transformants, there was no evidence for a clonotypic shift or acquired mutations in the expressed Ig V(H)DJ(H) gene segments, as further confirmed by a specific and sensitive PCR-single strand polymorphism analysis. In contrast, among the FL cells there was a high degree of intraclonal diversification with highly divergent V(H)DJ(H) gene sequences. Despite this intraclonal heterogeneity, the related DLBL expressed a collinear but unique V(H)DJ(H) gene sequence. The intraclonal genealogical tree for the FL case demonstrated that the DLBL emerged in association with unique V(H)DJ(H) gene mutational events. Among the intraclonal FL and related DLBL transformants, the nature and distribution of the Ig V(H)DJ(H) gene mutations were consistent with antigenic selection. Thus, clonal evolution in the transformation from low- to high-grade B cell lymphoma may involve distinct pathways which vary according to the cellular origin and the type of the progenitor B cell tumor.

Figure 1

Nucleotide sequences of the Ig V_HDJ_H genes expressed by the CLL (case 3557), SLL (case 1186) and FL (case 7473) and corresponding DLBL cells. In each case, the top sequence is provided for comparison and represents that of the closest reported germ-line V_H, D, or J_H gene. Dots indicate sequence identity, solid lines mark CDR, and numbers indicate the amino acid residues.

Figure 2

Deduced amino acid sequences of the Ig V_HDJ_H gene segments expressed by the CLL, SLL, FL and corresponding DLBL cells. Dots indicate identity and solid lines mark the CDR.

Figure 3

Genealogical tree outlining the clonal evolution of the FL case and transformed DLBL. Ig V_HDJ_H genes were sequenced using ten independent isolates from the FL tumor and from the associated DLBL. The ten sequences expressed in the FL tumor demonstrated considerable heterogeneity (FL-A, B, C, D, E, F, G, H, I, J), whereas the ten DLBL sequences were identical. By aligning these FL and DLBL V_HDJ_H gene segment sequences, a putative ancestor Ig gene sequence was deduced, and a genealogical tree was constructed. The common ancestor and putative intermediate clones are depicted as gray circles, and the number of mutations in each clone as compared to the closest ancestor is indicated. The 13 differences between the putative common ancestor and the first malignant cell would account for the mutations observed within the FL-A, B, C, and D elements that emerged in the third and fourth generations.

Figure 4

PCR-SSCP analysis of the expressed Ig V_H genes in the original CLL, SLL, and FL tumor and corresponding DLBL cells. cDNA corresponding to leader-J_H region of the expressed Ig V_HDJ_H gene segment were amplified in the presence of [α-³²P]dCTP, denatured, and separated by PAGE prior to autoradiography. In each case, non-denatured PCR products (ND) were used as controls, as indicated.

Figure 5

Detection of the the clone-specific Ig V_HDJ_H gene sequence in DLBL and FL cells of case 7473. DLBL cells of case 7473 were mixed with HL-60 cells in ratios of 1:10 × 1:10⁶. cDNA was prepared, and clone-specific primers were used to PCR amplify the specific DLBL Ig V_HDJ_H gene sequence. Electrophoretic fractionation of the PCR products in 2 % agarose containing 1 μ/ml ethidium bromide shows detection of the specific DLBL Ig V_HDJ_H gene sequence in 7374 DLBL cell “diluted” up to 10⁻⁴ to 10⁻⁵ in “irrelevant” cells, but not in the corresponding (“undiluted”) FL cells.