Search

The BCL6 gene encodes a POZ/Zinc-finger protein, which acts as a sequence-specific transcriptional repressor. It is expressed in B cells within the germinal centers (GC) and is required for GC formation. In approximately 40% of diffuse large cell lymphomas (DLCL) and approximately 14% of follicular lymphomas (FL), the BCL6 gene is rearranged by chromosomal translocations, which juxtapose heterologous promoters and 5' untranslated sequences derived from other chromosomes to the BCL6 coding domain or by mutations in the 5' regulatory region. To understand the functional consequence of the chromosomal translocations, we have studied the patterns of expression of the promoters found juxtaposed to BCL6 in DLCL and FL during B-lineage differentiation. Distinct heterologous 5' untranslated regions (IGH, IGL, TTF) were identified fused to the BCL6 coding domain by analysis of BCL6 cDNAs in two DLCL cases and one mixed follicular lymphoma (MxFL). These three sequences, as well as three other previously identified BCL6 fusion partners (IGHG3, BOB1, H4), were studied for their pattern of expression during B-lineage differentiation by Northern blot analysis of B-cell lines representation by Northern blot analysis of B-cell lines representative of the pre-B, B, immunoblast, and plasma cell stages. In contrast to BCL6, whose transcription is activated only in B cells within the GC, all of the other sequences displayed a broader pattern of expression ranging from constitutive expression throughout B-cell differentiation to persistent expression in immunoblasts and plasma cells. These results indicate that the expression of BCL6 is deregulated as a consequence of fusion to heterologous promoter regions. The persistent expression of activated BCL6 may contribute to lymphomagenesis by blocking B-cell differentiation within the GC.

The present study was designed to characterize the adhesive interaction of c-myc-transfected B-lymphoblastoid cell lines with resting and interleukin-1-activated endothelial cells. The transfected cell lines expressed lower levels of leukocyte function-associated antigen-1 compared with control non-transfected lines, while no reduction of expression of other surface structures, including the beta 1 integrin very late antigen-4, was observed. The transfected cell lines adhered to resting or activated endothelial cells less than control cells. Anti-CD18 monoclonal antibody inhibited binding of control cell lines but had a modest or no effect on adhesion of transfected cell lines. Anti-very late antigen-4 monoclonal antibody effectively inhibited binding of both transfected and control cell lines; this was more pronounced in the presence of anti-CD18, suggesting a cooperative interaction between these adhesion pathways. Transfected cell lines also had an impaired ability to penetrate endothelial cell monolayers in a transmigration assay. Our results indicate that activation of the c-myc oncogene in B-cells causes alterations in the adhesive interaction with endothelial cells. This may be relevant in the localization and malignant behavior of B-cell lymphomas carrying an activated c-myc oncogene.

Commonly observed in lymphoid neoplasms, deletions of 6q have been correlated with histologic and clinical subsets of non-Hodgkin's lymphoma (NHL). Our recent analysis of loss of heterozygosity of 6q loci in NHL showed two regions of minimal molecular deletion (RMD), an RMD1 at 6q25–27 and an RMD2 at 6q21–23. To establish correlations between these RMDs and regions of minimal cytogenetic deletions (RCDs) on 6q, and to define associations between RCDs and clinico-pathologic features, we have analyzed chromosome 6 abnormalities in 459 consecutively ascertained, karyotypically abnormal cases of NHL. Among these, 126 (27.5%) cases had structural abnormalities of chromosome 6, of which 94 were deletions. Analysis of these deletions identified three RCDs. An RCD1 encompassing 6q25–27 was seen in 45 intermediate- grade NHL. An RCD2 at 6q21 was observed in 11 high-grade NHL, 9 of which were of the immunoblastic subtype. An RCD3 at 6q23 was noted in 18 low-grade NHL lacking a t(14;18) translocation. Of these 18 cases, 12 were small lymphocytic NHL and, in 2 of these, del(6q) was the sole karyotypic abnormality. In 20 cases of low-grade NHL with t(14;18), the deletions spanned both RCD1 and RCD3. These data suggested the presence of at least 3 tumor suppressor genes on 6q within RCD1, RCD2, and RCD3; they also showed associations between RCDs in 6q and subsets of NHL, including a specific association between a group of well-differentiated lymphoid neoplasms and RCD3. The apparent heterogeneity of breakpoints when all NHLs are considered together explains the inability of previous studies to reliably establish correlations between recurring 6q deletions and histologic and clinical features of NHL.

The presence of the translocation t(8;14)(q24;q32) has not been well described in follicular lymphoma (FL). In a consecutive series of 278 karyotypically abnormal non-Hodgkin's lymphomas (NHL), six patients with FL showing a t(8;14) without a t(14;18)(q32;q21) were identified. They ranged in age from 45 to 73 years. The cell type was mixed in four patients, small-cleaved in one, and large-cleaved in one; four cases also contained diffuse areas. All cases tested displayed monoclonal surface Ig. The clinical courses were consistent with the histologic subtypes, being less aggressive than other t(8;14)-bearing NHL. In five cases, frozen tissue was available for Southern blotting. The BCL2 gene showed a germline configuration when studied with the MBR, MCR, and 5′ cDNA probes. The MYC gene also appeared unrearranged using an exon-1 probe with EcoRI or HindIII digestion. Analysis of the Ig heavy chain (IgH) gene with a JH region probe and BamHI or EcoRI digestion showed only one rearranged band in all cases, indicating that the 14q32 breakpoint did not lie in either the J or switch-mu (SM) regions. In four cases, the exon-1/intron-1 border of the MYC gene, a target area for point mutations in cases of t(8;14) that do not display rearrangements of the MYC gene, was enzymatically amplified and sequenced; no point mutations were identified. The indolent behavior of our six cases, and the finding that the molecular structure of the t(8;14) in these cases does not follow the pattern of breakpoint sites and point mutations defined in other histologic subtypes of NHL with this translocation, suggest that the t(8;14) in these cases is cytogenetically and molecularly distinct from the t(8;14) seen in high- grade NHLs, and is relatively ineffectual in terms of MYC deregulation, or that other genetic elements at these chromosomal sites may be involved. Further analysis of these tumors may provide insights into MYC deregulation and BCL2-independent FL.

For many decades, B cell chronic lymphocytic leukemia (B-CLL) stood out as a B cell-derived malignancy that was difficult to position within the framework of the available B cell differentiation scheme: First, the histology as well as the immunophenotype did not quite resemble that of any normal lymphocyte; second, in contrast to almost all other B cell tumor subtypes, the immunoglobulin variable region (IgV) genes of B-CLL cases could be either unmutated or somatically mutated; third, the genomic lesions observed in B-CLL were markedly distinct from those of the other major B cell malignancies, which typically exhibit balanced chromosome translocations. Recent advances in the characterization of both B-CLL and normal B cell subpopulations by phenotypic analysis, global gene expression profiling, as well as extensive IgV gene repertoire analyses have shed new light on the phenotype and the cell derivation of B-CLL and provided novel hypotheses concerning its pathogenesis. Here we summarize recent work relevant to these issues and conclude that B-CLL may be derived from a cell that can be referred to as a marginal zone B cell. Moreover, we propose that the lack of chromosomal translocations in B-CLL may be related to their derivation from marginal zone B cells, since somatic hypermutation and Ig class switch, the processes that generate chromosome translocations in most germinal center (GC)-derived malignancies, are no longer active in marginal zone B cells. Also, we discuss similarities and differences between B-CLL and hairy cell leukemia (HCL) and suggest that also HCL may be derived from a post-GC memory or marginal zone B cell.

The PTEN gene is involved in 10q23 deletions in several types of cancer, including glioma, melanoma, endometrial and prostate carcinomas. The PTEN gene product is a dual-specificity phosphatase with putative tumor suppressor function. Deletions and rearrangements of 10q22-25 have been reported in approximately 5%-10% of non-Hodgkin's lymphomas (NHLs), raising the possibility of PTEN involvement in these tumors. In order to address this question, we analyzed a panel of NHLs (n = 74) representative of the main histologic subtypes for mutations and homozygous deletions of PTEN. We report somatic coding/splice site mutations in 20% (2 of 10) of Burkitt's lymphoma cell lines and in 3% (2 of 64) of primary NHL cases analyzed. No homozygous deletions were found in these tumors. Interestingly, this study showed that cytogenetically characterized NHL cases (n = 6) with 10q22-q25 abnormalities displayed neither biallelic deletions nor mutations of PTEN. These results suggest that a tumor suppressor gene distinct from PTEN may be involved in 10q deletions in this subgroup of NHL cases.

Chromosomal band 3q27 frequently engages in translocations with a number of sites within the genome, including those containing IG and other genes, during the development of B-cell lymphoma. The BCL6 gene, mapped at 3q27, is deregulated in these translocations and was isolated from a t(3;14)(q27;q32) translocation. It encodes a zinc-finger transcription repressor protein, which is expressed mainly in the germinal center (GC) B cells and plays a key role in GC development and T-cell-dependent immune response. BCL6 deregulation in 3q27 translocations is brought about by substitution of its 5' regulatory sequences by promoters of the rearranging genes. BCL6-rearranging genes studied so far (IGH, IGLL, TTF, BOBI, H4) displayed a broader pattern of expression than BCL6 during B-cell development. This observation has led to the suggestion that continued expression of BCL6 beyond its developmentally regulated point of downregulation under the direction of substituted promoters may keep the GC B cell in a cycling mode and lead to clonal expansion and lymphoma development. However, the rearranging partners of BCL6 in several of the 3q27 translocations have not yet been identified. In a molecular cloning analysis of two such translocations, t(1;3)(q21;q27) and t(3;6)(q27;p25), and an immunoblastic lymphoma cell line, OSI-LY8, we identified a novel mechanism of BCL6 deregulation. This comprised replacement of BCL6 5' regulatory sequences by insertion of IG gene transcriptional regulatory sequences at the translocation junction. These studies demonstrate novel features of instability of 3q27, and of the BCL6 and IGH genes, in B-cell lymphomagenesis.

Chromosomal band 3q27 exhibits recurring and nonrecurring translocations and other rearrangements in approximately 8% of B-cell non-Hodgkin lymphomas (NHL) belonging to low-grade as well as diffuse aggressive histologies. The BCL6 gene, which encodes a zinc-finger transcription repressor protein and which maps to chromosomal band 3q27, is deregulated in t(3;14)(q27;q32) and other translocations by substitution of its transcription regulatory sequences by those of genes on the partner chromosomes. To delineate the cytogenetics and investigate the nature and consequence of BCL6 involvement in the spectrum of 3q27 aberrations seen in NHL, we analyzed a panel of 53 NHL tumors with 3q27 aberrations for BCL6 gene rearrangements and a subset of 32 of these for mutations. We identified four new recurring translocations involving 3q27, in addition to the previously recognized t(3;14)(q27;q32) and its variant, t(3;22)(q27;q11). Histologically, the 3q27 breaks were represented by 4% mantle cell lymphomas, 38% follicular center cell lymphomas, and 58% diffuse large B-cell lymphomas. Approximately 50% of the tumors exhibited BCL6 rearrangements, whereas 87.5% showed mutations in the 5' noncoding region which contains the transcription regulatory sequences. These results demonstrate that a substantial proportion of cytogenetically detected 3q27 breaks in NHLs do not represent BCL6-associated translocations. They also suggest alternate breakpoints which may lead to BCL6 deregulation, or involvement of other genes in 3q27 translocations. The frequent BCL6 mutation in these tumors is consistent with our previous observation of hypermutation of the 5' noncoding region of the gene in lymphomas arising in the germinal-center B-cells.

The t(9;14)(p13;q32) translocation is associated with approximately 50% of lymphoplasmacytoid lymphoma (LPL), a subtype of B-cell non-Hodgkin's lymphoma (NHL). We cloned the chromosomal breakpoint of der (14) from an LPL case (1052) and showed that it involved a junction between 9p13 and the switch micro region of the Ig heavy chain locus (IgH) on 14q32. Using a YAC contig spanning 1.5 megabase (Mb), we determined that the 9p13 breakpoint in one case (1052) mapped within a 270-kb restriction fragment containing two previously reported 9p breakpoints associated with a alpha-heavy chain disease case (MAL) and KI-1 positive diffuse large cell lymphoma (DLCL) cell line (KIS-1). The same fragment also contained the PAX-5 gene which encodes a B-cell specific transcription factor involved in the control of B-cell proliferation and differentiation. The breakpoints of KIS-1 and 1052 were mapped within the 5' noncoding region of PAX-5, while the 9p13 breakpoint of MAL mapped 230 to 270 kb upstream to PAX-5. In all three cases, the translocation caused the juxtaposition of the PAX-5 gene to the IgH locus in the opposite direction of transcription. When compared with six other DLCL cell lines lacking t(9;14)(p13;q32), the KIS-1 cell line showed an 11-fold overexpression of PAX-5 mRNA and a significantly reduced expression of the p53 gene, which is normally regulated by PAX-5. Moreover, metaphase and interphase fluorescence in situ hybridization (FISH) analysis using a YAC clone spanning 1 Mb including the PAX-5 as a probe identified chromosomal translocations in 5 of 7 cases carrying 9p13 translocations. These findings suggest that the PAX-5 gene is the target of the t(9;14) in LPL whereby its expression may be deregulated by juxtaposition to IgH regulatory elements, thus contributing to lymphomagenesis.

The identification of genes inducing resistance to anticancer chemotherapeutic agents and their introduction into hematopoietic cells represents a promising approach to overcome bone marrow toxicity, the limiting factor for most high-dose chemotherapy regimens. Because resistance to cyclophosphamide has been correlated with increased levels of expression of the aldehyde-dehydrogenase (ALDH1) gene in tumor cell lines in vitro, we tested whether ALDH1 overexpression could directly induce cyclophosphamide resistance. We have cloned a full-length human ALDH1 cDNA and used retroviral vectors to transduce it into human (U937) and murine (L1210) hematopoietic cell lines that were then tested for resistance to maphosphamide, an active analogue of cyclophosphamide. Overexpression of the ALDH1 gene resulted in a significant increases in cyclophosphamide resistance in transduced L1210 and U937 cells (50% inhibition concentration [IC50], approximately 13 mumol/L). The resistant phenotype was specifically caused by ALDH1 overexpression as shown by its reversion by disulfiram, a specific ALDH1 inhibitor. ALDH1 transduction into peripheral blood human hematopoietic progenitor cells also led to significant increases (4- to 10-fold; IC50, approximately 3 to 4 mumol/L) in cyclophosphamide resistance in an in vitro colony-forming assay. These findings indicate that ALDH1 overexpression is sufficient to induce cyclophosphamide resistance in vitro and provide a basis for testing the efficacy of ALDH1 gene transduction to protect bone marrow cells from high-dose cyclophosphamide in vivo.

Rearrangements of the NFKB2 gene are associated with lymphoid malignancies, but the functional significance of these alterations is not known. Here we characterize structurally and functionally a rearranged NFKB2 gene identified at the T cell lymphoma line, HUT78. The rearrangement has truncated NFKB2 sequences within the 3' ankyrin domain, leading to the production of truncated mRNA species and proteins as detected by Northern blot and immunoprecipitation analysis, respectively. Cloning and sequencing of the corresponding cDNAs indicates that, via alternative splicing, the rearranged gene codes for two proteins of 84 and 85 kD (p84/85) which retain the DNA-binding rel domain and the first five ankyrin repeats, but have lost their carboxy-terminus including the seventh ankyrin repeat. Immunofluorescence and immunoprecipitation analysis of HUT78 cells indicate that p84/85 are abnormally located in the nucleus in an unprocessed form, suggesting that these proteins can escape the cytoplasmic retention typical of the normal NFKB2 p100 protein before it is processed into p52. Electrophoretic mobility shift assays performed on HUT78 nuclear extracts indicate that the abnormal NFKB2 proteins bind kappa B sites specifically and alter the composition of NF-kappa B complexes in HUT78 cells. Transient co-transfection assays involving NFKB2 expression vectors and kappa B-driven reporter plasmids indicate that NFKB2 p85 has lost the transcriptional repressor functions typical of normal NFKB2 p52. These data indicate that the NFKB2 gene rearrangement detected in HUT78 cells leads to the production of abnormal NFKB2 proteins capable of altering the function of the NF-kappa B transcription system. Since analogous rearrangements are found in lymphoid malignancies, these findings further support a role of NFKB2 alterations in tumorigenesis.

The NFKB-2 gene (previously LYT-10, NF-kappa Bp100 or NF-kappa Bp97) codes for a NF-kappa B/rel related protein which is highly homologous to NFKB-1 (previously NF-kappa Bp105) within its rel, poly-glycine and ankyrin domains. The NFKB-2 gene is a candidate proto-oncogene since it is involved in lymphoma-associated chromosomal aberrations. In order to gain insight into the physiological function and role in tumorigenesis of NFKB-2, we have analysed its mechanism of expression and role in transcriptional regulation. We report that, contrary to previous studies, a single 3.2 kb mRNA species and its 100 kD (p100) primary translation product is detectable in all cell types tested. A second NFKB-2 protein, p52, corresponding to the amino-terminal half (rel domain) of NFKB-2 p100, is detectable in the same cell types and derives from the post-translational processing of p100. While p100 is constitutively localized in the cytoplasm, NF-kappa B induction by TPA treatment of Hela cells is associated with cytoplasmic/nuclear translocation of NFKB-2 p52 and its appearance within DNA-binding NF-kappa B complexes. NFKB-2 p52 differs from NFKB-1p50 in its differential affinity for kappa B sequences: by itself it binds H2/HLA-kappa B sites more efficiently than HIV/IgK-kappa B sites, while it can bind both sites efficiently when complexed with Rel-A(p65). Transient co-transfection of expression and reporter plasmids in cells devoid of endogenous NF-kappa B activity showed that p52 has no intrinsic transcriptional activation capabilities: it can stimulate Rel-A(p65)-driven transcription by formation of p65/p52 heterodimers, whereas, overexpressed, it down-regulates p65-dependent transcription by formation of inactive p52/p52 homodimers. These results indicate that the NFKB-2 gene codes for an inducible NF-kappa B transcription factor with the capability of differentially regulating NF-kappa B transcription depending on its abundance in the nucleus.

Chromosomal translocations involving band 3q27 and various chromosomal sites, including the sites of the immunoglobulin (Ig) loci (14q32, 2p12, 22q11), represent recurrent aberrations in non-Hodgkin's lymphoma (NHL). In order to identify the putative protooncogene involved in these translocations, we have cloned the breakpoints from two B-cell NHL cases carrying t(3;14)(q27;q32) translocations by screening genomic DNA libraries constructed from NHL biopsy samples with immunoglobulin probes. Several recombinant phages have been obtained from each case and shown to contain sequences from both 14q32 and 3q27 by fluorescence in situ hybridization mapping on metaphase chromosomes. In both cases, the translocation breakpoints were found within the switch region of the Ig heavy-chain locus on 14q32 and within the same 3-kilobase region on 3q27. When used in Southern blot hybridization, a probe from the 3q27 region detected rearrangements in an additional five NHL cases carrying 3q27 translocations with 14q32 or other genomic sites. The same probe detected a predominant 2.4-kilobase mRNA species in several lymphoid cell lines analyzed by Northern blot hybridization. These data suggest that chromosomal breakpoints in 3q27 cluster in the proximity of a transcribed gene which represents a candidate protooncogene (bcl-6) involved in B-cell NHL pathogenesis.

We have used fluorescence in situ hybridization (FISH) to perform precise chromosomal mapping of the genes encoding the transcription factors c-rel, NF-kappa Bp50, NF-kappa Bp65, and lyt-10. The previously published assignments of c-rel and NF-kappa Bp50 have been refined to specific bands. The map position of lyt-10, inferred from its isolation from a t(10;14)(q24;q32) translocation, has been confirmed. NF-kappa Bp65 has now been mapped to 11q13, a site of frequent involvement in aberration in multiple tumor types.

The Epstein-Barr virus (EBV) is associated with distinct forms of human lymphoid malignancies, including the endemic (eBL) and sporadic forms of Burkitt's lymphoma (sBL) and acquired immunodeficiency syndrome-associated non-Hodgkin lymphoma (AIDS-NHL). However, whether EBV has a pathogenetic role in these tumors or is a passenger virus has not been conclusively demonstrated. One element to distinguish between these two possibilities is to determine whether EBV infection has preceded and, thus, possibly contributed to clonal expansion, or whether infection has occurred after clonal expansion and thus is unlikely to contribute to pathogenesis. Toward this end we analyzed the structure of the heterogeneous genomic termini of EBV as markers of clonal infection in a panel of eBL (11 cases), sBL (9 cases), and AIDS-NHL (10 cases) biopsies. We show that EBV termini are uniformly clonal in sBL, eBL, and AIDS-NHL, strongly suggesting that EBV infection has preceded and, thus, most likely contributed to clonal expansion in these malignancies.

We previously identified a novel protein tyrosine kinase gene, tyk2, by screening a human lymphoid cDNA library with a tyrosine kinase domain specific c-fms restriction fragment under low stringency hybridization conditions. We have now isolated and sequenced a full length tyk2 cDNA clone; demonstrated that this gene is widely expressed in hematopoietic and non-hematopoietic cell lines; and mapped it to chromosome 19p13.2. The cDNA clone is 4176 nucleotides long and codes for a putative protein with a molecular weight of 134 kilodaltons. Hydrophobicity analysis of our sequence does not identify a transmembrane domain, which is found in all members of the receptor class of protein tyrosine kinases; nor can we detect an SH2 domain, found in all previously identified non-receptor protein kinases. We therefore propose that tyk2 is the prototype of a new class of non-receptor protein tyrosine kinases.

To identify novel protein tyrosine kinase (PTK) genes expressed in human lymphoid cells, we have screened B- and T-cell cDNA libraries at low stringency using a c-fms tyrosine kinase domain probe. Three new PTK genes were identified, based on the presence of conserved amino acid sequence motifs characteristic of the catalytic domain of tyrosine kinases. Of these three genes, one (tyk1) appears to be the human homologue of a previously cloned murine gene (ltk), which has been reported to encode a tyrosine kinase with a unique structure; while the second gene, tyk2 cannot be clearly assigned to any of the known PTK subfamilies, and therefore may be the prototype of a new PTK gene subfamily. The third gene (tyk3/fer) has been very recently cloned by others; we present additional characterization in this report. We have performed Northern blots to establish the size of the mRNA encoded for by these genes, and to confirm their expression in lymphoid cells. Finally, we have determined the chromosomal location of all three genes by analyzing human-mouse somatic cell hybrids.

We have determined the complete nucleotide sequence of human cellular c-myc, which is homologous to the transforming gene, v-myc, of myelocytomatosis virus MC29. Analysis of the genetic information and alignment with the known sequence of chicken c-myc and v-myc indicates: (i) An intervening sequence can be identified by consensus splice signals. The unique 5' sequence of c-myc and its junction with the v-myc region may be a canonical 3' splice acceptor. (ii) The c-myc locus can generate a mRNA whose termination signals are downstream from the translational termination signal. (iii) The three myc genes share the same reading frame, including translational termination signals. (iv) The homology is conserved only in the coding region. (v) Most changes at the nucleotide level result in no change in the amino acid. (vi) There are two distinct domains--the 5' unique domain, which is different from the viral, and the 3' coding domain, which contains amino acids coded by the two exons whose sequences have been determined here. In the latter domain, the amino acid variation between v-myc and chicken c-myc is less than 2%, whereas that between the chicken v-myc and the human is 27%, with the variation concentrated in the region that flanks the splicing points.

Ig and T beta gene rearrangements can be used as genetic markers of lineage and clonality in the study of B and T cell populations. We have addressed the issue of the respective B and T lineage specificity of these rearrangements by analyzing a panel of 63 lymphoid tumors representative of the various clinicopathologic categories of both B and T neoplasias. We report that approximately 10% of the cases tested displayed rearrangements of both Ig and T beta genes. Despite their dual genotypic pattern, these tumors retain a pure immunophenotype, i.e. they display either B or T cell lineage-restricted cell surface antigens. The implications of these findings for both normal and neoplastic lymphoid differentiation are discussed.

EVIDENCE for the involvement of more than one cell line and even interconversion of different forms of human leukaemia suggests that the neoplastic transformation occurs in a common stem cell1. Although erythroid leukaemias are very rare in humans, disturbances in haemopoiesis or haemoglobin synthesis2 are common in leukaemia, suggesting the possible involvement of erythroid precursors. We show here, by the use of sensitive cDNA (complementary DNA)–RNA hybridisation, that in a substantial number of human leukaemias globin RNA sequences can be demonstrated in nuclear RNA prepared from highly purified leukaemic cell populations.

Splenic marginal zone lymphoma (SMZL) is a B cell malignancy of unknown pathogenesis, and thus an orphan of targeted therapies. By integrating whole-exome sequencing and copy-number analysis, we show that the SMZL exome carries at least 30 nonsilent gene alterations. Mutations in NOTCH2, a gene required for marginal-zone (MZ) B cell development, represent the most frequent lesion in SMZL, accounting for ∼20% of cases. All NOTCH2 mutations are predicted to cause impaired degradation of the NOTCH2 protein by eliminating the C-terminal PEST domain, which is required for proteasomal recruitment. Among indolent B cell lymphoproliferative disorders, NOTCH2 mutations are restricted to SMZL, thus representing a potential diagnostic marker for this lymphoma type. In addition to NOTCH2, other modulators or members of the NOTCH pathway are recurrently targeted by genetic lesions in SMZL; these include NOTCH1, SPEN, and DTX1. We also noted mutations in other signaling pathways normally involved in MZ B cell development, suggesting that deregulation of MZ B cell development pathways plays a role in the pathogenesis of ∼60% SMZL. These findings have direct implications for the treatment of SMZL patients, given the availability of drugs that can target NOTCH, NF-κB, and other pathways deregulated in this disease.

Activation of the c-myc oncogene has been implicated in the pathogenesis of T-cell malignancies in species other than man [1–3]. In order to establish a possible involvement of this oncogene in human T-cell neoplasias, we investigated the c-myc structure in several primary T-cell tumors as well as in several leukemic T-cell lines. The Hut 78 line, derived from a Sezary syndrome patient, was found to have a c-myc rearrangement beginning immediately 3′ to c-myc exon 3 [4]. The abnormal c-myc also appears to be duplicated compared to the normal allele. Chromosome analysis reveals that trisomy is the only cytogenetic anomaly involving chromosome 8, suggesting that the duplicated chromosome is the one carrying the abnormal c-myc and ruling out a Burkitt’s type translocation event.

The frequency and pattern of T gamma gene rearrangement and expression was investigated in hematopoietic neoplasms including T and B lymphoid and myeloid malignancies. 39 of 39 T lymphoid neoplasms, including fresh cases and cell lines, were found to display clonal T gamma gene rearrangements. There was heterogeneity with respect to utilization of the two T gamma constant region genes, T gamma C1 and T gamma C2. In 31 cases (80%) T gamma C1 was deleted and T gamma C2 was rearranged, while in the remaining 8 cases (20%) T gamma C1 was rearranged. T gamma gene rearrangements were found in non-T cells, but were restricted to 6/17 (35%) immature B cell neoplasms. All 24 mature B cell and 14 myeloid neoplasms retained the T gamma germ line pattern. T gamma mRNA was found in all T cells tested. However, the majority (16/17) of T cells most likely do not express a T gamma protein since a T alpha/beta heterodimer detected by reactivity with the MoAb WT31 is present on the cell surface together with T3. These data suggest that T gamma gene rearrangements are universal in T cells and frequent in immature B cell neoplastic populations. However, expression of the T gamma protein is extremely infrequent, indicating that T cell neoplasms are very rarely derived from the recently identified T3+T gamma +T alpha/beta- peripheral T cell population.

The frequent occurrence of Kaposi's sarcoma (KS) in association with the acquired immune deficiency syndrome (AIDS) could be due to the fact that the etiological agent of this tumor is the same retrovirus causing AIDS, to another oncogenic virus frequently found in AIDS patients, or to the unmasking of the tumorigenic potential of KS cells by immunosuppression. We have therefore investigated the presence of DNA sequences homologous to the AIDS retrovirus, cytomegalovirus (CMV), and hepatitis B virus in 13 KS necropsies and biopsies from AIDS patients. All KS DNA samples were negative for AIDS retrovirus or hepatitis B DNA sequences. Two DNAs from necropsies contained CMV DNA, but the data suggested the presence of replicating CMV DNA due to generalized infection. We have also studied cell cultures derived from KS skin biopsies of AIDS patients. These cultures had a short lifetime in vitro and expressed some markers of endothelial cells. The cells were not tumorigenic in nude mice but contained a number of chromosomal rearrangements which were often monoclonal within the same culture. However, these abnormalities were different from culture to culture and even in cultures from the same biopsy. The presence of these chromosomal abnormalities seemed to correlate with the cell positivity for endothelial markers. Taken together these results indicate that neither the AIDS retrovirus, CMV, or hepatitis B virus is directly responsible for the altered growth of KS cells, that KS may be polyclonal even within the same lesion, and that KS cells have a tendency to karyotypic rearrangements.

We investigated the organization of the immunoglobulin heavy chain (IgH) and the T cell receptor beta chain (T beta) gene loci in 20 ocular adnexal and four extraocular lymphoid neoplasms obtained from 18 patients presenting with an ocular adnexal lymphoid neoplasm. Fifteen ocular adnexal and four extraocular lymphoid neoplasms occurring in 13 patients were classified by morphological examination and immunophenotypic analysis as monoclonal B cell lymphomas. Each one of these 19 lymphoid neoplasms exhibited clonal IgH gene rearrangements upon hybridization of EcoRI- or HindIII-digested DNA to a heavy-chain joining region (JH)-specific DNA probe. The bilateral ocular adnexal monoclonal B cell neoplasms occurring simultaneously in two individuals exhibited identical clonal IgH gene rearrangements, which indicated their derivation from an identical B cell clone. The ocular adnexal and the extraocular monoclonal B cell neoplasms occurring in two of three patients also exhibited identical clonal IgH gene rearrangements, which suggested that they too were derived from an identical B cell clone. Five ocular adnexal lymphoid neoplasms were classified by morphological examination and immunophenotypic analysis as benign, polyclonal pseudolymphomas. Three of these five ocular adnexal lymphoid neoplasms exhibited clonal IgH gene rearrangements, which suggested the presence of monoclonal B cell populations that escaped detection by morphological and immunophenotypic examination. None of the 24 pathological samples exhibited clonal T beta gene rearrangements upon hybridization of EcoRI- or BamHI-digested DNA to a T beta gene DNA probe. The results of these studies demonstrate the value of Southern blot hybridization analysis for clonal IgH and T beta gene rearrangements in the diagnosis, classification, and investigation of extranodal lymphoid neoplasms originating and/or presenting in the ocular adnexa.

Human follicular B-cell lymphomas possess a t(14;18) that translocates a putative protooncogene, BCL2, into the immunoglobulin heavy chain locus. The normal BCL2 gene is quiescent in resting B cells, expressed in proliferating, but down-regulated in differentiated B cells. Inappropriately high levels of BCL2-immunoglobulin chimeric RNA are present in t(14;18) lymphomas for their mature B-cell stage. We examined the biologic effects of BCL2 deregulation in human B cells by introducing BCL2 into human B-lymphoblastoid cell lines (LCLs) with retroviral gene transfer. Although deregulated BCL2 expression as a single agent was not sufficient to confer tumorigenicity to LCLs, it consistently produced a 3- to 4-fold increment in LCL clonogenicity in soft agar. In addition, BCL2 deregulation complements the transforming effects of the MYC oncogene in LCLs. BCL2 augmented the clonogenicity of LCLs bearing exogenous MYC and increased the frequency and shortened the latency of tumor induction in immunodeficient mice. These results demonstrate a role for BCL2 as a protooncogene that affects B-cell growth and enhances B-cell neoplasia.

We have shown that 1. partial provirus integration can be a possible result of a natural infection, and may serve as a model in animal systems where a viral etiology is implicated but detection of a major fraction of the virus genome is rare; 2. All human DNA contains some sequences that hybridize specifically with genomes of SiSV-SiSAV, suggesting that viruses of this group have infected humans in the past and recombined with human cellular DNA. 3. Finally, DNA from uncultured leukocytes of two leukemic patients, one being HL23, which yielded the virus HL23V in culture, was shown to have virus specific fragments related to BaEV. Another human DNA sample revealed virus specific fragments related to SiSV(SiSAV). These fragments are probably acquired by infection.

A patient with T-cell acute lymphoblastic leukemia (T-ALL) in second remission was treated with high doses of chemotherapy and radiotherapy, followed by transplantation of autologous bone marrow purged ex-vivo with an anti-CD5-saporin immunotoxin (OKT1-SAP). Prior to transplantation the bone marrow graft had been considered in complete remission, as assessed by morphology and immunophenotyping. Twenty-two days after transplantation, the disease relapsed in the bone marrow with the same phenotype as at the onset. Retrospective analysis of the transplanted marrow cells by a recently developed high sensitivity and specificity assay (HSS assay), involving immunologic fractionation and T-cell receptor rearrangement analysis, revealed a graft contamination of approximately 0.5% malignant T-cells. This finding, together with the early post-transplant leukemic relapse, strongly suggests that the bone marrow was the source of the leukemic cells. The data are discussed for their implications on residual leukemia detection by gene rearrangement studies.

The large granular lymphocyte (LGL) population, which effects a natural killer (NK) function, consists of cells whose lineage derivation has not been clearly established on the basis of phenotypic and functional properties. To clarify the relationship of LGL/NK cells to T cells we studied patterns of rearrangement and expression of the T cell receptor (Ti) genes alpha, beta, and gamma in normal human LGLs; in CD8+, CD8-, Mol+, and Mol- LGL subsets; and in 17 cases of leukemic LGL proliferations (T gamma LPD). T alpha, T beta, and T gamma genes were not expressed, nor were T beta and T gamma genes rearranged in normal LGLs or LGL subsets. The T gamma LPD were divided into two groups. One group (15/17 cases) was characterized as CD3+ and displayed Ti gene rearrangements. Seven of these cases were reactive with monoclonal antibody WT31, which suggested expression of an alpha/beta heterodimer on the cell surface. The other group (2/17 cases) was CD3- with unrearranged Ti genes. These results indicate that the normal LGL/NK population is homogeneous and distinct from the normal T cell population because it does not express, and as a result, cannot effect its immune function through the T cell receptor molecules. Conversely, T gamma LPDs represent a heterogeneous group of lymphoproliferative diseases within which the CD3-, Ti- cases most likely represent the neoplastic counterpart of normal LGL cells. The more frequent CD3+ cases may be related to recently described NK-like T cells. The observations that normal LGLs maintain germline T gamma genes and that many CD3+ T gamma LPD display an alpha/beta heterodimer suggest that a T gamma-containing receptor may not be necessary for NK or NK-like cytotoxicity.

We investigated the rearrangement patterns of the gene coding for the beta chain of the T cell receptor (T beta) in 11 patients with T-cell derived chronic lymphoproliferative disorders, including T-cell prolymphocytic leukemia (T-PLL) and T-cell chronic lymphocytic leukemia (T-CLL). We found that all five cases of T-PLL, and five of six cases of T-CLL, displayed T beta-gene rearrangements, clearly establishing their monoclonal nature. Clonality could not be determined in one case of T-CLL where the T beta gene was found unrearranged. Our results demonstrate that the majority of cases of both clinically aggressive T-PLL and clinically indolent T-CLL are monoclonal. These results suggest that the analysis of T beta gene rearrangements represents a valid tool for the differential diagnosis and clinical monitoring of T-cell derived chronic lymphoproliferative diseases.

Type C retroviruses are associated with naturally occurring leukemia-lymphomas in many animal species, including man (see Gallo et al., this volume), and they are also the first tangible tools for approaches to our understanding of the molecular mechanisms of cellular transformation (see Dues-berg et al. and Vande Woude et al., this volume). While most retroviruses isolated in nature are slow acting in disease induction (chronic leukemia viruses), a subclass of viruses, including the sarcoma viruses and acute leukemia viruses, cause disease rapidly in vivo and transform appropriate target cells efficiently in vitro. These properties are conferred on the viruses by a viral transforming (v-onc) gene. There are now at least 17 different v-onc genes identified in retroviruses isolated from avian, rodent, feline, and primate species. All viral onc genes are derived from normal cellular genes (c-onc genes) of their host of origin. C-onc genes share several common features: (1) they are highly conserved among all vertebrates and some are conserved even in nonvertebrate species. For example, sequences related to a few onc genes have been identified in Drosophila [8], and an enzyme related to pp60 src of Rous sarcoma virus has been detected in sponge (M. Schartl, personal communication). (2) With few exceptions, the homology between c-onc and v-onc genes is interrupted by nonhomologous stretches in c-onc, tentatively referred to as introns. (3) Most c-onc genes have been found to be expressed at least at some stages of normal cell growth, suggesting they are functional genes in normal cellular processes.

The association between certain human tumours and characteristic chromosomal abnormalities has led to the hypothesis that specific cellular oncogenes may be involved and consequently 'activated' in these genetic recombinations. This hypothesis has found strong support in the recent findings that some cellular homologues of retroviral onc genes are located in chromosomal segments which are affected by specific tumour-related abnormalities (see ref. 4 for review). In the case of human undifferentiated B-cell lymphoma (UBL) and mouse plasmacytomas, cytogenetic and chromosomal mapping data have identified characteristic chromosomal recombinations directly involving different immunoglobulin genes and the c-myc oncogene (for review see refs 5, 6). In UBLs carrying the t(8:14) translocation it has been shown that the human c-myc gene is located on the region of chromosome 8 (8q24) which is translocated to the immunoglobulin heavy-chain locus (IHC) on chromosome 14. Although it is known that the chromosomal breakpoints can be variably located within or outside the c-myc locus and within the IHC mu (refs 9, 11) or IHC gamma locus, the recombination sites have not been exactly identified and mapped in relation to the functional domains of these loci. We report here the identification and characterization of two reciprocal recombination sites between c-myc and IHC mu in a Burkitt lymphoma. Nucleotide sequencing of the cross-over point joining chromosomes 8 and 14 on chromosome 14q--shows that the onc gene is interrupted within its first intron and joined to the heavy-chain mu switch region. This recombination predicts that the translocated onc gene would code for a rearranged mRNA but a normal c-myc polypeptide.

Viral transforming (v-onc) genes are derived from cellular (c-onc) genes that are highly conserved among vertebrates. Comparative studies of v-onc and c-onc genes have shed some light on the mechanism leading to formation of the transforming viruses. A specific example of the sis gene is presented here for illustration. Studies on the expression of six c-onc genes in human cells revealed at least three categories of onc genes: (a) those that are universally expressed and probably are important in basic cellular functions, (b) those that are not detectably expressed in the cells examined and may have very transient expression in development, and (c) those that are only expressed in specific cell types and may be important in tissue differentiation. Our studies do not show conclusively a role of these onc genes in human neoplasias.

There have been reports of sporadic findings of type-C markers in human cells. Most commonly these markers were related to the two classes of primate type-C viruses: The simian sarcoma virus-simian sarcoma associated virus complex [SiSV (SiSAV)] and gibbon ape leukemia virus (GaLV) group and the baboon endogenous virus, BaEV (see Gallo elsewhere in this book). However, detection of proviral sequences related to either of these virus groups was rare, and a preliminary survey indicated that DNA from leukemic tissues, although hybridizing more viral probe (SiSAV and BaEV) than DNA from normal tissues, displayed a broad range of hybridization values which never approached the level of DNA from virus-infected tissue culture cells (Gallo, elsewhere in this book). One possible explanation for the low but possibly significant hybridization values found in fresh leukemic cells is that subgenomic fragments could have been integrated in the DNA of these tissues rather than complete provirus. However, unequivocal evidence for partial provirus integration of type-C RNA viruses is still lacking in the literature. In this report we show by restriction enzyme-blotting analysis, that (i) a few tissues from a gibbon ape exposed to GaLV contained an incomplete provirus; (ii) DNA from all human DNA contained sequences that hybridize specifically to SiSV-SiSAV genomes, suggesting a recombination event between these viruses and human DNA via infection; (iii) DNA from two leukemia DNA samples showed extra, presumably acquired, viral fragments related to BaEV.

Sixteen cases of histologic intermediate-grade and high-grade AIDS-associated non-Hodgkin's lymphoma (NHL) were studied for the presence and patterns of c-myc gene and bcl-2 locus rearrangements. The presence of Epstein-Barr virus (EBV) sequences and proteins and HTLV-I sequences were also investigated. c-myc gene rearrangements analogous to those observed in sporadic Burkitt lymphomas were detected in 12 of 16 cases. Six of 16 cases had detectable EBV sequences and proteins. None of the cases displayed bcl-2 rearrangements or contained HTLV-I sequences. These data suggest a frequent role for c-myc activation in the pathogenesis of AIDS-associated NHL, independent of histologic type. Conversely, EBV does not appear to be directly involved in lymphomagenesis in the majority of AIDS-associated NHLs.