High frequency of somatic mutations in the VH genes expressed in prolymphocytic leukemia

Prolymphocytic teukemia (PLL] is a chronic lymphoproliferative disorder, characterized by prominent splenomegaly, prolymphocytes accounting for more than 55% of circulating lymphocytes, and short-term survival. To better characterize the nature of the cellular origin in this disease, we analyzed Ig heavy chain variable region (V,) genes in eleven cases of de novo PLL. Leukemic cells expressed a skewed repertoire characterized by predominant use of the V3 family members (73%). with preferential use of the V3-23 gene (50% of the VH3 genes). All sequences from expressed Vn genes diverged from their putative germline counterpart, and in eight cases ROLYMPHOCYTIC leukemia (PLL) is a relatively rare P form of chronic lymphoproliferative disease, first described by Galton in 1974.' According to French-AmericanBritish (FAB) diagnostic criteria, the hallmark of the disease is the presence of more than 55% circulating morphologically identified prolymphocytes.' It is a distinct clinicopathological entity characterized by massive splenomegaly without lymph node enlargement, frequent marked hyperleukocytosis (often >lo0 x 109/L; the majority of which are prolymphocytes), and associated anemia and thrombopenia. Morphologically, prolymphocytes are large cells with condensed nuclear chromatin and a prominent central nucleolus. The vast majority of PLL are from the B-lineage origin and express high density monotypic surface immunoglobulin (sIg), usually IgM with or without IgD, and the CD19, CD20, and CD22 B cell marker^.^ In contrast to chronic lymphocytic leukemia (CLL), they often express FMC7, but rarely CD23. About half of the cases express the CD5 antigen. Although PLL is considered to be distinct from CLL by many criteria, the delineation between the two is not clearcut because some cases of CLL can transform into PLL: Furthermore, mixed forms of CLLPL have been identified in which the number of prolymphocytes is between 10% and 55%.5 Analysis of Ig genes has provided important information in understanding the biology of B-cell tumors6 After completing a series of complex DNA rearrangements of the Ig genes in the bone marrow, virgin B cells emigrate to the lymphoid organs. Further maturation of B lymphocytes occurs within the germinal centers of these organs, including the introduction of point mutations in the Ig variable (V) regions by the poorly understood mechanism of somatic hypermutation.' On stimulation by local antigens, those cells expressing V genes with mutations leading to higher affinity are selected and expand preferentially. This process results in the generation of memory B cells and high-affinity antibody producing cells. Consequently point mutations are found preferentially in nucleotides coding for amino acid residues critical for antigen-antibody interactions, the complementarity determining regions (CDR). In contrast, polyclonal stimulation not mediated by antigen results in mutations that are randomly distributed throughout the V regions.8 Analysis of V region nucleotide sequences can therefore the divergence was greater than 5%. In seven cases, which expressed the V3-23 gene and V,,4 family members, nucleotide substitutions could be confidently attributed to somatic mutations. The type and distribution of these mutations clearly indicated that in three cases the cells had been subjected to an antigen selection process. Taken together, these results suggest that B-PLL cells display a skewed repertoire of Ig VH regions and probably represent, at least in some instances, expansion of postgerminal center cells that have undergone antigen driven selection.