Adenoviral vectors efficiently target cell lines derived from selected lymphocytic malignancies, including anaplastic large cell lymphoma and Hodgkin's disease.

We have hypothesized that adenoviral vectors might mediate gene transfer into cell lines derived from human lymphocytic malignancies, such as lymphoma, lymphocytic leukemia, and myeloma. A panel of 33 cell lines was studied for their ability to be transduced by an adenoviral (AD) vector encoding the Escherichia coli beta-galactosidase gene (AD-betagal). A cytochemical assay and a flow cytometry assay both demonstrated that a subset of lymphocytic cell lines can be efficiently transduced by adenoviral vectors. In particular, three of three anaplastic large cell lymphoma lines, two of two Hodgkin's disease cell lines, two of seven Burkitt's lymphoma cell lines, and three of five myeloma cell lines exhibited efficient gene transfer. The ability of an AD vector expressing the thymidine kinase (tk) gene from herpes simplex virus-1 (AD-tk) followed by ganciclovir (GCV) to kill 11 of these lymphocytic cell lines was studied. In eight of the cell lines tested, more than 68% of the cells were killed by AD-tk/GCV. Similar results were obtained using an adenoviral vector expressing the wild-type p53 tumor suppressor gene (AD-p53). Thus, AD-tk/GCV and AD-p53 both demonstrated efficient killing of these cell lines. These data document that adenoviral vectors are valuable reagents for the introduction of genes into selected lymphocytic cell lines. These data also suggest that adenoviral vectors might be useful for gene therapy of subsets of lymphocytic malignancy.

Selective elimination (purging) of contaminating malignant cells from hematopoietic stem cell autografts using recombinant adenovirus.

In this work we have explored the use of adenoviral vectors for the purging of cancer cells from hematopoietic stem cell (HSC) autografts. We showed that a recombinant adenovirus expressing the herpes simplex-1 thymidine kinase gene (AD-tk) plus ganciclovir (GCV) killed HELA cells more effectively than did GCV alone. HELA cells were then mixed with human HSCs and exposed to AD-tk/GCV. AD-tk/GCV reduced the number of HELA colonies to 4% of control values, with no detectable reduction in the hematopoietic progenitor, colony forming unit-granulocyte/monocyte (CFU-GM). Similar studies of the JB6 non-Hodgkins lymphoma cell line showed a reduction to 5% of controls; studies of MCF-7, a breast carcinoma cell line, showed a reduction to 30% of controls, with no CFU-GM toxicity. Thus, AD-tk mediated selective killing of contaminating tumor cells. We also evaluated a recombinant adenovirus encoding the tumor suppressor gene p53 (AD-p53). AD-p53 was able to selectively kill all three cell lines (reducing tumor colonies approximately 100-fold) without any toxicity to CFU-GM. Although both AD-tk/GCV and AD-p53 were effective in these experiments, AD-p53 seemed to be more potent. Adenoviral vectors show promise for selectively targeting cancer cells that contaminate HSC autografts.

Influence of molecular characteristics on clinical outcome in human immunodeficiency virus-associated non-Hodgkin's lymphoma: identification of a subgroup with favorable clinical outcome.

The relationship between clinical and molecular characteristics of 45 treated individuals with histologically-documented human immunodeficiency virus (HIV)-associated B-cell non-Hodgkin's lymphoma was examined to determine whether differences in molecular features of lymphoma were associated with differences in clinical outcome. Tissue specimens from these tumors were evaluated for evidence of Ig heavy-chain gene rearrangements using both Southern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Lymphomas were also evaluated for the presence of Epstein-Barr virus (EBV) DNA sequences and c-myc gene rearrangements. Twenty-five lymphomas were characterized as polyclonal and 20 as monoclonal. PCR amplification of expressed Ig variable (V)-region genes confirmed polyclonality in three extensively studied polyclonal lymphomas. The median CD4 count was significantly higher in the group with polyclonal disease (277/microL) than in the group with monoclonal disease (123/microL), P = .04. The complete response rate to therapy was significantly higher in patients with polyclonal disease (78%) and CD4 greater than 200/microL (81%) than in those with monoclonal disease (31%) and CD4 less than 200/microL (33%). CD4 count, clonality, and presence of EBV DNA sequences were the most important predictors of survival. Both Kaplan-Meier and Cox proportional hazards analyses showed a markedly prolonged survival in those patients with both CD4 > or = 200/microL and polyclonal disease. Histologically the polyclonal lymphomas were high grade in appearance and contained prominent macrophages. All seven surviving patients were in this group. Median survival for those individuals whose tumors contained EBV sequences was only 3.2 months (range, 0.4 to 19.5), whereas those with EBV- tumors survived for a median of 9.0 months (range, 0.7 to 65.2), P = .0007. These data indicate that molecular features of HIV-associated lymphomas may be important predictors of clinical outcome. These characteristics define a distinct subset of patients with polyclonal EBV- tumors and CD4 counts greater than 200/microL that appear to have a less aggressive clinical course.

Antigen expression and polymerase chain reaction amplification of mantle cell lymphomas.

Flow immunophenotyping, DNA content analysis, and polymerase chain reaction (PCR) amplification for t(11;14) and t(14;18) were performed on 11 cases of typical mantle cell lymphoma (MCL), 5 cases of apparent MCL with proliferation centers (MCL-PC), and 5 cases of small lymphocytic lymphoma (SLL). Immunophenotyping showed IgM (P < .001), Ig light (P < .001), and CD20 (P < .001) expression to be more intense in MCL than in SLL. In MCL-PC, the mean intensity of IgM, Ig light chain, and CD20 expression was intermediate to the intensities observed in MCL and SLL. Furthermore, in contrast to SLL, all MCL and 4 of 5 MCL-PC cases exhibited stronger CD20 than CD19 expression. CD10 expression was not observed in any case and CD5 expression was present in all SLL and MCL-PC cases and in 9 of 11 MCL cases. DNA content analysis showed an S-phase fraction of less than 3% in all cases studied and, except for 1 MCL case, all lymphomas were DNA diploid. The t(11;14) breakpoint junctions involving the bcl-1 major translocation cluster were amplified by PCR in 4 of 11 (36%) MCL cases and in none of the MCL-PC or SLL cases. The t(14;18) involving the bcl-2 major breakpoint region was not identified by PCR in any case. We conclude that the level of expression of surface antigens and the rapid detection of t(11;14) by PCR are potentially useful for distinguishing MCL and SLL in the clinical setting. Further investigations as to the biologic relationship between MCL, MCL-PC, and SLL, and the utility of t(11;14) PCR in these lymphomas are warranted.

Chromosome t(11;14)(q13;q32) breakpoints in centrocytic lymphoma are highly localized at the bcl-1 major translocation cluster.

Chromosome 11q13 translocation breakpoints have been found dispersed over more than 100 kb of genomic DNA in centrocytic lymphoma and lymphocytic lymphoma of intermediate differentiation (mantle cell lymphoma). Approximately one-half of these translocations occur at the bcl-1 major translocation cluster (MTC) and appear tightly clustered by Southern blot restriction mapping. In order to specifically characterize these t(11;14)(q13;q32) translocations, six cases of centrocytic lymphoma with MTC rearrangements on Southern blot were studied. Genomic DNA was amplified by PCR (polymerase chain reaction) using a consensus immunoglobulin heavy-chain joining gene (JH) primer and two separate MTC primers. Sequencing of the PCR products revealed the MTC breakpoints in all six cases to be clustered within a 61 basepair span, very similar to those previously reported in t(11;14)-containing human B-cell lines. No two breakpoints were identical. JH breakpoints involved JH4 in five cases and JH6 in the other. Tight clustering of the MTC breakpoints thus permits PCR detection of the t(11;14) in many cases of centrocytic lymphoma, which may be of value in classifying this subtype of non-Hodgkin's lymphoma.

A radiation hybrid map of the distal short arm of human chromosome 11, containing the Beckwith-Wiedemann and associated embryonal tumor disease loci.

We describe a high-resolution radiation hybrid (RH) map of the distal short arm of human chromosome 11 containing the Beckwith-Wiedemann gene and the associated embryonal tumor disease loci. Thirteen human 11p15 genes and 17 new anonymous probes were mapped by a statistical analysis of the cosegregation of markers in 102 rodent-human radiation hybrids retaining fragments of human chromosome 11. The 17 anonymous probes were generated from lambda phage containing human 11p15.5 inserts, by using ALU-PCR. A comprehensive map of all 30 loci and a framework map of nine clusters of loci ordered at odds of 1,000:1 were constructed by a multipoint maximum-likelihood approach by using the computer program RHMAP. This RH map localizes one new gene to chromosome 11p15 (WEE1), provides more precise order information for several 11p15 genes (CTSD, H19, HPX, ST5, RNH, and SMPD1), confirms previous map orders for other 11p15 genes (CALCA, PTH, HBBC, TH, HRAS, and DRD4), and maps 17 new anonymous probes within the 11p15.5 region. This RH map should prove useful in better defining the positions of the Beckwith-Wiedemann and associated embryonal tumor disease-gene loci.

Deletion of IRF-1, mapping to chromosome 5q31.1, in human leukemia and preleukemic myelodysplasia.

One of the most frequent cytogenetic abnormalities in human leukemia and myelodysplasia is an interstitial deletion within chromosome 5q. A tumor suppressor gene has been hypothesized to lie in 5q31, the smallest commonly deleted region. IRF-1, a gene whose product manifests anti-oncogenic activity, was mapped to 5q31.1. IRF-1 lies between IL-5 and CDC25C and is centromeric to IL-3 and GM-CSF. Among these genes, only IRF-1 was consistently deleted at one or both alleles in 13 cases of leukemia or myelodysplasia with aberrations of 5q31. Inactivating rearrangements of one IRF-1 allele, accompanied by deletion of the second allele, were also identified in one case of acute leukemia. Thus, IRF-1 may be a critically deleted gene in human leukemia and myelodysplasia.

Amplification and expression of the bcl-1 gene in human solid tumor cell lines.

The bcl-1 gene maps to chromosome 11q13 and has recently been shown to be a member of the cyclin gene family. Amplification of the chromosome region containing bcl-1 occurs frequently in breast cancer, squamous cell cancer, and other tumor types. We have hypothesized that amplification results in altered expression of the bcl-1 gene, contributing to carcinogenesis. In this work, we studied bcl-1 gene amplification and expression in a panel of human cell line. bcl-1 is expressed in all cell lines studied. The level of expression tends to be higher in amplified cell lines. We also screened these cell lines for int-2 and hst-1 expression, genes which are frequently coamplified with bcl-1. No int-2 expression was detected, and the two cell lines expressing hst-1 were unamplified. Our data provide support for the importance of bcl-1 in carcinogenesis.

A radiation hybrid map of the proximal long arm of human chromosome 11 containing the multiple endocrine neoplasia type 1 (MEN-1) and bcl-1 disease loci.

We describe a high-resolution radiation hybrid map of the proximal long arm of human chromosome 11 containing the bcl-1 and multiple endocrine neoplasia type 1 (MEN-1) disease gene loci. We used X-ray irradiation and cell fusion to generate a panel of 102 hamster-human somatic cell hybrids containing fragments of human chromosome 11. Sixteen human loci in the 11q12-13 region were mapped by statistical analysis of the cosegregation of markers in these radiation hybrids. The most likely order for these loci is C1NH-OSBP-(CD5/CD20)-PGA-FTH1-COX8-PYGM -SEA-KRN1-(MTC/P11EH/HSTF1/INT2)-GST3- PPP1A. Our localization of the human protooncogene SEA between PYGM and INT2, two markers that flank MEN-1, suggests SEA as a potential candidate for the MEN-1 locus. We map two mitogenic fibroblast growth factor genes, HSTF1 and INT2, close to bcl-1, a mapping that is consistent with previously published data. Our map places the human leukocyte antigen genes CD5 and CD20 far from the bcl-1 locus, indicating that CD5 and CD20 expression is unlikely to be altered by bcl-1 rearrangements. PPP1A, which has been postulated as a MEN-1 candidate tumor suppressor gene, and GST3, a gene transcriptionally active in many human cancers, both map distal to the bcl-1 translocation cluster and the region containing MEN-1, and therefore are unlikely to be directly involved in bcl-1 or MEN-1.

Characterization of a candidate bcl-1 gene.

The t(11;14)(q13;q32) translocation has been associated with human B-lymphocytic malignancy. Several examples of this translocation have been cloned, documenting that this abnormality joins the immunoglobulin heavy-chain gene to the bcl-1 locus on chromosome 11. However, the identification of the bcl-1 gene, a putative dominant oncogene, has been elusive. In this work, we have isolated genomic clones covering 120 kb of the bcl-1 locus. Probes from the region of an HpaII-tiny-fragment island identified a candidate bcl-1 gene. cDNAs representing the bcl-1 mRNA were cloned from three cell lines, two with the translocation. The deduced amino acid sequence from these clones showed bcl-1 to be a member of the cyclin gene family. In addition, our analysis of expression of bcl-1 in an extensive panel of human cell lines showed it to be widely expressed except in lymphoid or myeloid lineages. This observation may provide a molecular basis for distinct modes of cell cycle control in different mammalian tissues. Activation of the bcl-1 gene may be oncogenic by directly altering progression through the cell cycle.

Cloning of the t(11;14)(q13;q32) translocation breakpoints from two human leukemia cell lines.

The t(11;14)(q13;q32) translocation has been associated with several subtypes of human leukemia and lymphoma. It has been proposed that this translocation activates a proto-oncogene designated BCL1. In an effort to better understand the mechanism by which this translocation leads to malignancy, we have studied this translocation in two human cell lines. MO1094 and MO2058 were derived from patients with prolymphocytic variants of chronic lymphocytic leukemia. Southern blotting of the MO2058 cell line documented that the translocation linked the Jh region in the immunoglobulin heavy chain gene to the previously described BCL1 major translocation cluster (MTC). Using the polymerase chain reaction, we cloned this translocation and showed that the chromosome 11 breakpoint was within 7 bp of two other samples reported previously. Southern blotting of the MO1094 cell line suggested that the translocation in this cell line might link Jh sequences to a new region in the BCL1 locus on chromosome 11. Therefore, the MO1094 breakpoint was cloned from a genomic library. Comparison with normal cloned DNA from the BCL1 locus showed that the chromosome 11 breakpoint occurred 24 kb telomeric of the MTC. This work reinforces the concept that translocation breakpoints in the BCL1 locus are scattered over at least 63 kb.

Rearrangement of the chromosome 11 bcl-1 locus in centrocytic lymphoma: analysis with multiple breakpoint probes.

Centrocytic lymphoma is a B-cell non-Hodgkin's lymphoma (NHL) composed of lymphocytes resembling cleaved follicular center cells (centrocytes). Previous studies have suggested an association between t(11;14) chromosomal translocations and bcl-1 rearrangement in centrocytic and related intermediate lymphocytic lymphomas. To further characterize the association between bcl-1 and centrocytic lymphoma, Southern blot analysis was performed on samples from 23 patients using four separate bcl-1 breakpoint probes spanning 63 kb of the chromosome 11 bcl-1 locus. Rearrangements were identified in six patients with the major translocation cluster (MTC) probe and in another six with probe p94PS, located about 24 kb 5' of MTC. Eleven of these 12 cases showed comigration of rearranged bcl-1 and Ig heavy chain-joining genes, consistent with the t(11;14) chromosomal translocation. No rearrangements were observed with the bcl-1 locus probes p210 or p11EH located 5' of p94PS, nor with bcl-2 or c-myc oncogene probes. No bcl-1 rearrangements were identified in B-cell follicular NHL (15), small noncleaved cell (Burkitt's and non-Burkitt's) NHL (8), T-cell NHL (4), multiple myeloma (14), and pre-B-cell acute lymphoblastic leukemia (9). One of 23 B-cell NHL of large cell type and one of 19 chronic lymphocytic leukemias or small lymphocytic NHL had MTC rearrangement. Thus, bcl-1 rearrangement occurred at MTC or p94PS in 12 of 23 centrocytic lymphomas (52%), confirming a nonrandom association and suggesting a pathogenetic role for the bcl-1 locus in this immunohistologic subtype of NHL.

Evidence for molecular subtypes of HIV-associated lymphoma: division into peripheral monoclonal, polyclonal and central nervous system lymphoma.

The pathogenesis of the HIV-associated lymphomas is not well understood. In order to begin characterizing this class of lymphoma, we initiated a molecular genetic study of DNA extracted from 31 diagnostic biopsy specimens from patients diagnosed with AIDS-associated non-Hodgkin's lymphoma. Analysis of 25 peripheral lymphomas showed that 14 were monoclonal B-cell processes, while 11 appeared to be of polyclonal origin. Five of the 14 monoclonal lymphomas were found to have rearrangements of the c-myc gene. Epstein-Barr virus (EBV) genomes were found in seven out of 14 monoclonal samples, but only two out of nine polyclonal samples. The six primary central nervous system (CNS) lymphoma samples were more homogeneous than the peripheral samples and all were monoclonal, positive for EBV and lacked detectable c-myc gene rearrangements. This study allows us to subdivide the HIV-associated lymphomas into three major molecular subtypes: (1) monoclonal B-cell process frequently associated with c-myc rearrangement or detectable EBV genomes, (2) polyclonal B-cell process typically without evidence of EBV, and (3) monoclonal primary CNS process associated with EBV genomes and lacking detectable c-myc rearrangement.

AIDS-associated polyclonal lymphoma: identification of a new HIV-associated disease process.

High-grade non-Hodgkins B-cell lymphoma is one of the principle malignancies that occurs in individuals infected with the human immunodeficiency virus (HIV-1). Immunoblastic lymphomas that arise in immunosuppressed transplant patients have been described as both monoclonal and polyclonal, and occur in association with Epstein-Barr virus (EBV) infection. To test whether polyclonal lymphoma occurred in patients with AIDS we studied tumors from multiple sites in three patients who died with widespread AIDS-associated large cell or large cell immunoblastic lymphoma. All biopsy specimens contained invasive lymphoma. Tumor cells were mature IgM-positive immunoblasts by immunohistochemical analysis, with the same B-cell phenotype observed in all tumor sites. Only a minority of sites from all patients analyzed were monoclonal as measured by immunoglobulin gene rearrangements, with one case having several foci of monoclonal disease with other histologically identical metastases showing no evidence of monoclonal proliferation. Similar to the transplant-associated polyclonal B-cell proliferations. EBV gene sequences were present in multiple sites from one autopsy. In the other two autopsies, polyclonal B-cell proliferations occurred in the absence of EBV involvement except at one site, where a minor clone of EBV-infected cells was found. In contrast to HIV-associated Burkitt's lymphoma, no c-myc rearrangements were found at any site. These studies describe the occurrence of polyclonal lymphoma in AIDS and suggest that EBV-negative polyclonal lymphoma may be a distinct disease entity unique to HIV-infected individuals.

Primary central nervous system lymphomas: natural history and response to radiation therapy in 55 patients with acquired immunodeficiency syndrome.

The incidence of primary central nervous system (CNS) lymphoma has increased rapidly in patients with acquired immunodeficiency syndrome (AIDS) and is predicted to exceed 1800 cases annually by 1991. To characterize the natural history and response to radiation therapy (RT) of these lesions, the authors have reviewed the clinical histories of 55 AIDS patients with biopsy-proven primary CNS lymphomas. The tumors responded both clinically and radiologically to whole-brain RT consisting of 4000 rad in 267-rad fractions over 3 weeks or an equivalent neuroret dose. The mean duration of survival from the appearance of symptoms consistent with the mass lesion was significantly greater in patients who received RT than in those who did not (42 vs. 134 days, p less than 0.5; median 27 vs. 119 days). Autopsy findings showed that patients who did not receive RT died from tumor progression, whereas those who completed RT died of opportunistic infections. Patients with AIDS who are suspected of having primary CNS lymphoma should therefore immediately undergo biopsy and, if the diagnosis is confirmed, whole-brain RT. With early diagnosis and treatment, these tumors respond to, and patients benefit from, RT. Survival of such patients may in future be prolonged by more effective treatments for systemic opportunistic infections.

Activation of the interleukin-3 gene by chromosome translocation in acute lymphocytic leukemia with eosinophilia.

The t(5;14)(q31;q32) translocation from B-lineage acute lymphocytic leukemia with eosinophilia has been cloned from two leukemia samples. In both cases, this translocation joined the IgH gene and the interleukin-3 (IL-3) gene. In one patient, excess IL-3 mRNA was produced by the leukemic cells. In the second patient, serum IL-3 levels were measured and shown to correlate with disease activity. There was no evidence of excess granulocyte/macrophage colony stimulating factor (GM-CSF) or IL-5 expression. Our data support the formulation that this subtype of leukemia may arise in part because of a chromosome translocation that activates the IL-3 gene, resulting in autocrine and paracrine growth effects.

The human Pim-1 gene is selectively transcribed in different hemato-lymphoid cell lines in spite of a G + C-rich housekeeping promoter.

The expression of the Pim-1 proto-oncogene was studied by using the K562, Daudi, and Jurkat cell lines. In K562, Pim-1 mRNA levels were more than 20-fold higher than in Daudi and 50-fold higher than in Jurkat. Nuclear run-on assay data correlated directly with the steady-state mRNA levels, suggesting that the rate of transcription was responsible for the selective expression of this gene. Furthermore, the half-life of Pim-1 mRNA was shown to be 47 min in K562, 71 min in Daudi, and 35 min in Jurkat. This indicated that selective Pim-1 mRNA expression did not depend on posttranscriptional regulation. Therefore, 1.7 kilobases of the Pim-1 promoter was sequenced and studied in detail. The sequence showed that the region from nucleotide -1 to -873 was G + C rich (71%). Study of promoter deletions defined two major functional regions, a proximal element (nucleotide -104 to -1) and a distal element (nucleotide -427 to -336). DNase I protection assays identified binding sites for the Sp1 and AP2 proteins in these elements. A possible new transcription factor binds at position -348 in the distal element. In our study of the 1.7-kilobase Pim-1 promoter, we found no differences between K562 and Jurkat that could explain large differences in transcription. Therefore, the Pim-1 promoter appears to function constitutively, and we conclude that distant elements must regulate the tissue-selective expression of this gene. Although the Pim-1 gene has a G + C-rich housekeeping promoter, expression is carefully regulated at the level of transcription.

An additional breakpoint region in the BCL-1 locus associated with the t(11;14)(q13;q32) translocation of B-lymphocytic malignancy.

The t(11;14)(q13;q32) translocation is associated with human B-lymphocytic malignancy. This translocation divides the IgH locus on chromosome 14q32 and may activate a postulated proto-oncogene, bcl-1, located on chromosome 11q13. Two samples of chronic lymphocytic leukemia with the t(11;14)(q32;q13) translocation were studied. The break in one sample was shown to join Jh sequences with the previously described bcl-1 major translocation cluster. DNA blots of the second sample suggested that Jh sequences were joined to a different breakpoint region on chromosome 11. This translocation was cloned and found to link the human Jh3 region and a new breakpoint region 63 kb telomeric of the major translocation cluster. This translocation occurred in part as the result of an aberrant D-J recombination. Recurrent translocations human B-lymphocytic malignancy. The definition of a new breakpoint region may aid the identification of the postulated bcl-1 gene.

The t(5;14) chromosomal translocation in a case of acute lymphocytic leukemia joins the interleukin-3 gene to the immunoglobulin heavy chain gene.

Chromosomal translocations have proven to be important markers of the genetic abnormalities central to the pathogenesis of cancer. By cloning chromosomal breakpoints one can identify activated proto-oncogenes. We have studied a case of B-lineage acute lymphocytic leukemia (ALL) that was associated with peripheral blood eosinophilia. The chromosomal translocation t(5;14) (q31;q32) from this sample was cloned and studied at the molecular level. This translocation joined the immunoglobulin heavy chain joining (Jh) region to the promotor region of the interleukin-3 (IL-3) gene in opposite transcriptional orientations. The data suggest that activation of the IL-3 gene by the enhancer of the immunoglobulin heavy chain gene may play a central role in the pathogenesis of this leukemia and the associated eosinophilia.