Ajay Bakhshi

Immunoglobulin-Gene Rearrangements as Unique Clonal Markers in Human Lymphoid Neoplasms

Immunoglobulin genes in their germ-line form are separated DNA subsegments that must be joined by means of recombinations during B-cell development. Individual immunoglobulin-gene rearrangements are specific for a given B cell and its progeny. We show that the detection of such gene rearrangements by Southern hybridization provides a sensitive marker for both clonality and B-cell lineage within lymphoid tissues lacking expression of definitive surface phenotypes. We have used these genetic markers in three ways: to establish a diagnosis of lymphoma in a neoplastic disorder of uncertain cell type, to show that some lymphomas that were previously classified as being of T-cell type in fact contain monoclonal B cells, and to detect clonal B-cell populations within lymphomatous tissues of uncertain immunotype and within an atypical lymphofollicular hyperplasia having no other clonal surface markers. These sensitive and unique indicators of clonality located directly at the DNA level are capable of providing insights into the cellular origin, early detection, and natural history of neoplasia.

Lymphoid Blast Crises of Chronic Myelogenous Leukemia Represent Stages in the Development of B-Cell Precursors

The origin and stage of differentiation of the blast-crisis cells in chronic myelogenous leukemia have remained uncertain. Because immunoglobulin heavy-chain and light-chain genes must undergo a DNA rearrangement during B-cell development but rarely do so in human non-B-cell lineages, we examined these genes in 18 episodes of chronic myelogenous leukemia. In eight of nine episodes of lymphoid blast crisis, heavy-chain genes were rearranged, and in three, rearrangements in light-chain genes were also present. In contrast, cells from chronic myeloid, myeloid blast, and erythroid-like phases retained germ-like immunoglobulin genes. The observed phenotypic markers and gene configurations revealed that most lymphoid blast crises represent stages of development of B-cell precursors. In two separate episodes of lymphoid crisis, cells from a single patient possessed identical heavy-chain but different light-chain-gene configurations. Thus, the precursor cells that monoclonally expand to produce a lymphoid crisis are capable of immunoglobulin-gene rearrangements and represent discrete steps in early B-cell maturation.

Gene rearrangements as markers of clonal variation and minimal residual disease in acute lymphoblastic leukemia.

Immunoglobulin (Ig) heavy (H) and light (L) chain gene rearrangements were used as molecular markers of clonal evolution and minimal residual disease in B cell precursor acute lymphoblastic leukemia (ALL). All leukemic episodes within individual patients shared at least one identical Ig rearrangement and thus arose from a common clonal progenitor cell. Nine of 11 patients displayed completely identical patterns between leukemic episodes, while two of 11 patients demonstrated genetic progression between diagnosis and relapse as evidenced by additional rearrangements. These genetic changes marked the emergence of leukemic subclones. Ig gene rearrangements were also used as sensitive markers to identify clonal cell populations in ALL patients following induction or reinduction therapy and to search for residual bone marrow disease in patients in clinical remission or with isolated extramedullary relapse. DNA rearrangements provide tumor-specific markers to follow the genetic variation of ALL and may facilitate the early detection of recurrent disease.

Molecular Genetic Analysis of Human Lymphoid Neoplasms: Immunoglobulin Genes and the c-myc Oncogene

Immunoglobulin genes responsible for individual antibodies are organized as discontinuous DNA segments in their germline form. As an uncommitted stem cell develops into an antibody-synthesizing plasma cell, rearrangements of these immunoglobulin gene segments serve to activate the genes and to generate the virtually unlimited capacity to synthesize antibodies that recognize potential antigens. The analysis of immunoglobulin gene structure and arrangement has been of immense value in the study of human lymphoid neoplasms. Recombinant DNA technology involving analysis of immunoglobulin gene arrangement has been used to classify neoplasms of previously uncertain lineage, aid in the diagnosis of neoplasms of the B-cell series, and define the state of differentiation of neoplastic B-cell precursors. Furthermore, the demonstration of translocation of a particular transforming gene, the c-myc oncogene, into the immunoglobulin gene locus in Burkitts lymphoma has provided a major insight into the cause of malignant transformation of these lymphoid cells.Abstract Immunoglobulin genes responsible for individual antibodies are organized as discontinuous DNA segments in their germline form. As an uncommitted stem cell develops into an antibody-synthes...

In vitro enhancement of immunoglobulin gene expression in chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (CLL) cells appear to be arrested in their differentiation so that little immunoglobulin is secreted in most cases. To determine their capacity for further differentiation we stimulated cells from a series of 10 cases of CLL with a phorbol ester and assayed for production of immunoglobulin protein, accumulation of immunoglobulin mRNA, and alterations in cell surface markers. We found that cells from all cases were induced to secret monoclonal immunoglobulin of the same heavy and light chain type as the surface membrane immunoglobulin type. Immunoglobulin secretion was preceded by a rapid increase in the levels of mRNA coding for IgM, predominantly the secretory form, mu s-mRNA, rather than the membrane form, mu m-mRNA. A similar selection of mu s- over mu m-mRNA is known to occur in plasma cells by a mechanism of differential processing of mRNA from a single mu-chain gene. Except for a decline in the expression of surface IgD, cell surface determinants remained unaffected both in terms of the percentage of positive cells and the relative number of sites per cell. In contrast to previous studies, these results indicate that CLL cells consistently retain the capacity to further differentiate toward plasma cells and secrete immunoglobulin. The immunoglobulin secretion is mediated, at least in part, by a developmentally regulated increment in mu s-mRNA.

Immunoglobulin chain gene rearrangements in a t(4;ll) acute leukaemia with monocytoid blasts

Summary. We report a case of acute leukaemia with the t(4;l 1) chromosomal translocation which, at initial diagnosis, had L‐l lymphoblasts that were positive for terminal deoxynucleotidyl transferase (TdT) and HLA‐DR but negative for myeloid cytochemical markers. At last relapse the patient had mostly monocytoid blasts which were now TdT negative but were positive for HLA‐DR, weakly positive for Sudan Black B (SB), periodic acid Schiffs (PAS), naphthol AS‐D acetate esterase (NSE), chloroacetate esterase (CAE) and negative for acid phosphatase (AP) and nitroblue tetrazolium (NBT) reduction. Treatment with 12‐o‐tetradecanoylphorbol‐13‐acetate (TPA) in vitro induced differentiation to macrophage‐Iike cells that were strongly positive for SB, PAS, NSE, AP, CAE and NBT reduction, indicating a latent monocyte‐like phenotype. Thus the leukaemic cell clone or a precurser clone with the t(4;l 1) translocation manifested a lymphoid phenotype at initial diagnosis and a monocytoid phenotype at relapse. Immunoglobulin gene analysis of the monocytoid relapse blasts revealed rearrangements of the heavy chain gene alleles and germline light chain genes. Thus, the leukaemia clone with the t(4;ll) chromosomal translocation could be a bipotential cell with heavy chain gene rearrangements occurring in a primitive cell which may retain the ability to differentiate along the myeloid‐monocytoid lineage in response to the appropriate stimulus. Alternatively, these characteristics may result from a transformation associated event.

Genetic Rearrangements of Human Immunoglobulin Genes

The immunoglobulin (Ig) genes ultimately responsible for the generation of antibody diversity and thus the uniqueness or idiotype of each individual molecule produced are organized in a subsegmental, discontinuous fashion in their germ-line form.(1–4) As we will explore, the design of these coding gene subsegments and even the probable mechanisms for their DNA assemblage have been remarkably conserved over evolutionary time.(5–9) This elaborate system utilizes movable gene subsegments, flexibility at the sites of their recombination, and somatic mutation to maximize the number of unique antibody molecules that can be generated from a necessarily limited amount of germline material. While the somatic process of gene recombination creates an amazing diversity of products, these rearrangements have also proven to be rather error-prone. Multiple, alternative chances and choices for assembling an Ig gene appear to compensate for these abortive attempts. We will show that the assemblage of Ig gene subsegments occurs in a sequential fashion during early B-cell development and helps ensure that the final B cell makes but a single Ig molecule. This hierarchy of Ig gene recombination events, mandatory during B-cell development, has proved of great importance in assessing the clonality and stage of differentiation of a variety of human neoplasms. Following the rearrangements that generate the antibody diversity and idiotypes necessary for immune regulation, the Ig gene loci may at times undergo an additional recombination. As we will see, this latter rearrangement is one between chromosomes and results in the translocation of a cellular oncogene to an Ig gene locus within certain human B-cell malignancies.

Molecular Markers of Clonality, Lineage, Differentiation, and Translocation in B Cell Neoplasms

The demonstration that distinct types of lymphoid neoplasms could be assigned to stages of B or T cell development provided great insights into the biology of these malignancies1,2. Historically this has been approached by utilizing cell surface markers associated with various developmental stages of B or T cell maturation. Despite a large number of lineage-associated cell surface markers, it is still frequently impossible to conclusively classify a lymphoid neoplasm as B or T cell in origin. This is often due to the admixture of large numbers of nonneoplastic cells with the neoplastic cells in a lymphomatous tissue. Alternatively, other malignancies may be at a stage of differentiation prior to the expression of any lineage-restricted cell surface antigen. Moreover, the determination of clonality in lymphoid neoplasms has for all practical purposes been limited to the mature B cell malignancies that display the presence of but one light (L)-chain isotype, κ or λ. The DNA rearrangements which assemble the gene subsegments for antigen-specific receptors in B cells as well as T cells serve as molecular markers which are unique to individual neoplasms. The rearranging determinants have made an enormous contribution to refining the diagnosis and understanding the pathogenesis of lymphoid neoplasms.