Timothy W. McKeithan

The candidate proto-oncogene bcl-3 is related to genes implicated in cell lineage determination and cell cycle control

A gene, bcl-3, is found on chromosome 19 adjacent to the breakpoints in the translocation t(14;19)(q32;q13.1), which occurs in some cases of chronic lymphocytic leukemia. Sequence analysis of the human bcl-3 gene predicts a protein containing seven tandem copies of the SWI6/cdc10 motif. This motif was previously identified in yeast genes that regulate events at the start of the cell cycle and in invertebrate transmembrane proteins involved in cell differentiation pathways. Expression of bcl-3 in normal blood cells increases markedly following mitogenic stimulation, and leukemic cells with the translocation show much greater expression than controls. These results suggest that bcl-3 is a proto-oncogene that may contribute to leukemogenesis when abnormally expressed.

Molecular signatures to improve diagnosis in peripheral T-cell lymphoma and prognostication in angioimmunoblastic T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is often challenging to diagnose and classify. Gene expression profiling was performed on 144 cases of PTCL and natural killer cell lymphoma and robust molecular classifiers were constructed for angioimmunoblastic T-cell lymphoma (AITL), anaplastic lymphoma kinase-positive (ALK(+)) anaplastic large-cell lymphoma (ALCL), and adult T-cell leukemia/lymphoma. PTCL-unclassifiable was molecularly heterogeneous, but we were able to identify a molecular subgroup with features of cytotoxic T lymphocytes and a poor survival compared with the remaining PTCL-not otherwise specified cases. Many of the pathologic features and substantial components of the molecular signature of AITL are contributed by the follicular dendritic cells, B-cell, and other stromal components. The expression of Th17-associated molecules in ALK(+) ALCL was noted and may represent aberrant activation of Th17-cell differentiation by abnormal cytokine secretion. Adult T-cell leukemia/lymphoma has a homogeneous molecular signature demonstrating high expression of human T-lymphotropic virus type 1-induced genes. These classifiers reflect the biology of the tumor cells as well as their microenvironment. We also constructed a molecular prognosticator for AITL that appears to be largely related to the microenvironmental signature, and the high expression of 2 immunosuppressive signatures are associated with poor outcome. Oncogenic pathways and tumor-host interactions also were identified, and these findings may lead to better therapies and outcome in the future.

Morphology in Ki-1(CD30)-positive non-Hodgkin's lymphoma is correlated with clinical features and the presence of a unique chromosomal abnormality, t(2;5)(p23;q35).

Ten patients with strongly Ki-1(CD30)-positive non-Hodgkins lymphoma (NHL) were identified at our institution during the past 5 years. Based on morphology, the lymphomas of five of these patients were classified as anaplastic large-cell lymphoma (ALCL); the lymphomas of four patients lacked the morphologic features of ALCL (non-ALCL); and the lymphoma of one patient was unclassifiable. Significant clinical and cytogenetic differences were observed between patients with ALCL and those with non-ALCL. The patients with ALCL tended to be young at the time of diagnosis. They presented with peripheral lymphadenopathy, and two of the five patients had skin involvement. An identical reciprocal translocation involving chromosomes 2 and 5 [t(2;5)(p23;q35)] was observed in lymph nodes from each of the two ALCL patients whose chromosomes were studied. Four of the five patients with ALCL are alive and in complete remission 10–27 months after receiving systemic chemotherapy. In contrast, the patients with non-ALCL were heterogeneous with respect to clinical findings. All of the non-ALCLs were histologically aggressive; however, their morphology varied. The t(2;5) was absent in the lymphoma specimens from each of three non-ALCL patients studied. Three of the four patients died within 17 months after receiving systemic chemotherapy. Thus, differences in morphology are correlated with differences in the clinical findings, karyotype, and outcome in Ki-1-positive NHL.

Distinctive patterns of BCL6 molecular alterations and their functional consequences in different subgroups of diffuse large B-cell lymphoma.

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal (PM) DLBCL. The BCL6 gene is often translocated and/or mutated in DLBCL. Therefore, we examined the BCL6 molecular alterations in these DLBCL subgroups, and their impact on BCL6 expression and BCL6 target gene repression. BCL6 translocations at the major breakpoint region (MBR) were detected in 25 (18.8%) of 133 DLBCL cases, with a higher frequency in the PM (33%) and ABC (24%) subgroups than in the GCB (10%) subgroup. Translocations at the alternative breakpoint region (ABR) were detected in five (6.4%) of 78 DLBCL cases, with three cases in ABC and one case each in the GCB and the unclassifiable subgroups. The translocated cases involved IgH and non-IgH partners in about equal frequency and were not associated with different levels of BCL6 mRNA and protein expression. BCL6 mutations were detected in 61% of DLBCL cases, with a significantly higher frequency in the GCB and PM subgroups (>70%) than in the ABC subgroup (44%). Exon-1 mutations were mostly observed in the GCB subgroup. The repression of known BCL6 target genes correlated with the level of BCL6 mRNA and protein expression in GCB and ABC subgroups but not with BCL6 translocation and intronic mutations. No clear inverse correlation between BCL6 expression and p53 expression was observed. Patients with higher BCL6 mRNA or protein expression had a significantly better overall survival. The biological role of BCL6 in translocated cases where repression of known target genes is not demonstrated is intriguing and warrants further investigation.

The miRNA-17∼92 cluster mediates chemoresistance and enhances tumor growth in mantle cell lymphoma via PI3K/AKT pathway activation.

The median survival of patients with mantle cell lymphoma (MCL) ranges from 3 to 5 years with current chemotherapeutic regimens. A common secondary genomic alteration detected in MCL is chromosome 13q31-q32 gain/amplification, which targets a microRNA (miRNA) cluster, miR-17∼92. On the basis of gene expression profiling, we found that high level expression of C13orf25, the primary transcript from which these miRNAs are processed, was associated with poorer survival in patients with MCL (P=0.021). We demonstrated that the protein phosphatase PHLPP2, an important negative regulator of the PI3K/AKT pathway, was a direct target of miR-17∼92 miRNAs, in addition to PTEN and BIM. These proteins were down-modulated in MCL cells with overexpression of the miR-17∼92 cluster. Overexpression of miR-17∼92 activated the PI3K/AKT pathway and inhibited chemotherapy-induced apoptosis in MCL cell lines. Conversely, inhibition of miR-17∼92 expression suppressed the PI3K/AKT pathway and inhibited tumor growth in a xenograft MCL mouse model. Targeting the miR-17∼92 cluster may therefore provide a novel therapeutic approach for patients with MCL.

Activating mutations of STAT5B and STAT3 in lymphomas derived from γδ-T or NK cells

Lymphomas arising from NK or γδ-T cells are very aggressive diseases and little is known regarding their pathogenesis. Here we report frequent activating mutations of STAT3 and STAT5B in NK/T-cell lymphomas (n=51), γδ-T-cell lymphomas (n=43) and their cell lines (n=9) through next generation and/or Sanger sequencing. STAT5B N642H is particularly frequent in all forms of γδ-T-cell lymphomas. STAT3 and STAT5B mutations are associated with increased phosphorylated protein and a growth advantage to transduced cell lines or normal NK cells. Growth-promoting activity of the mutants can be partially inhibited by a JAK1/2 inhibitor. Molecular modelling and surface plasmon resonance measurements of the N642H mutant indicate a marked increase in binding affinity of the phosphotyrosine-Y699 with the mutant histidine. This is associated with the prolonged persistence of the mutant phosphoSTAT5B and marked increase of binding to target sites. Our findings suggest that JAK-STAT pathway inhibition may represent a therapeutic strategy.

BCL3 rearrangements and t(14;19) in chronic lymphocytic leukemia and other B-cell malignancies: A molecular and cytogenetic study

The t(14;19)(q32.3;q13.1) is a recurring translocation found in the neoplastic cells of some patients with chronic lymphocytic leukemia (CLL) or other B‐lymphocytic neoplasms. We previously cloned the translocation breakpoint junctions present in the leukemic cells from three such patients and identified a gene, BCL3, whose transcription is increased as a result of the translocation. In the present paper, we describe three additional patients with the t(14;19), one with lymphoma and two with CLL, and report the cloning and sequencing of the breakpoint junction in one of these patients as well as in a previously reported patient. We and others have found that the breakpoints on chromosome 14, with one exception, fall within the switch region upstream of the immunoglobulin heavy chain Cα1 or Cα2 sequences. Several of the breaks within chromosome 19 fall immediately upstream of the BCL3 gene, but several others are more than 16 kb 5′ of the gene. Most patients with CLL and the t(14;19) also show trisomy 12. Genes Chromosom. Cancer 20:64–72, 1997.

The human programmed cell death-2 (PDCD2) gene is a target of BCL6 repression: Implications for a role of BCL6 in the down-regulation of apoptosis

BCL6, a gene on chromosome 3 band q27, encodes a Kruppel-type zinc finger transcriptional repressor. Rearrangements of this gene are frequent in various kinds of lymphomas, particularly of the large-cell B-cell type. The BCL6 nuclear phosphoprotein is expressed in a variety of tissues and is up-regulated particularly in lymph node germinal centers. The zinc fingers of BCL6 bind DNA in a sequence-specific manner. To identify targets of the BCL6 repressive effects, we used a VP16-BCL6 fusion protein containing the zinc fingers but devoid of the repressor domains to compete with the binding of endogenous BCL6 in a transiently transfected B-cell line and then performed subtractive hybridization by using a method to selectively amplify sequences that are differentially expressed. We found that the programmed cell death-2 (PDCD2) gene is a target of BCL6 repression. This gene is the human homolog of Rp8, a rat gene associated with programmed cell death in thymocytes. Immunohistochemistry reveals the anticipated inverse relationship between BCL6 and PDCD2 expression in human tonsil. PDCD2 is detectable in cells of the germinal center in areas where there is less BCL6 expression as well as in the mantle zone, where there is little or no BCL6 expression. These results raise the possibility that BCL6 may regulate apoptosis by means of its repressive effects on PDCD2. BCL6 deregulation may lead to persistent down-regulation of PDCD2, reduced apoptosis, and, as a consequence, accumulation of BCL6-containing lymphoma cells.

Live-Cell Dynamics and the Role of Costimulation in Immunological Synapse Formation

Using transfected fibroblasts expressing both wild-type I-Ek and green fluorescent protein-tagged I-Ek with covalently attached antigenic peptide, we have monitored movement of specific MHC:peptide complexes during CD4+ T cell-APC interactions by live-cell video microscopy. Ag recognition occurs within 30 s of T cell-APC contact, as shown by a sharp increase in cytoplasmic calcium ion concentration. Within 1 min, small MHC:peptide clusters form in the contact zone that coalesce into an immunological synapse over 3–20 min. When T cells conjugated to APC move across the APC surface, they appear to drag the synapse with them. This system was used to examine the role of costimulation in the formation of the immunological synapse. Blocking CD80/CD28 or ICAM-1/LFA-1 interactions alters synapse morphology and reduces the area and density of accumulated complexes. These reductions correlate with reduced T cell proliferation, while CD69 and CD25 expression and TCR down-modulation remain unaffected. Thus, costimulation is essential for normal mature immunological synapse formation.

Resolution of Helicobacter pylori-associated gastric lymphoproliferative disease in a child

A possible causative association between Helicobacter pylori infection and gastric lymphoproliferative disorders has recently been recognized. The case of a 14-year-old girl who was diagnosed with H. pylori gastritis and associated gastric lymphoproliferative disease of the low-grade mucosa-associated lymphoid tissue type is reported. The patient was treated only for the H. pylori infection (amoxicillin, bismuth, and metronidazole) without any adjuvant chemotherapy or surgery for her lymphoproliferative disorder. This treatment not only resulted in the eradication of the microorganism but also complete resolution of her lymphoproliferative disease. The patient was subsequently followed up for a period of 7 years. There has been no histological recurrence of H. pylori gastritis or gastric lymphoproliferative disease. It is believed that this is the first report to describe a long-term follow-up of an H. pylori-associated gastric lymphoproliferative disorder in a pediatric patient who was exclusively treated for H. pylori infection. The observations in this report suggest that H. pylori-associated low-grade gastric lymphoproliferative disease can be completely cured by eradicating the organism. Therefore, this therapeutic approach, combined with close follow-up, should be the treatment of choice in children with this associated condition before attempting more aggressive treatments, thus potentially avoiding chemotherapy and/or (partial) gastrectomy.