Mechanisms of Ly2 suppressor cell activity. Activation of an Ly1 I-J+ cell is required to transduce the suppressive signal.

A cell-free product secreted by Ly1-2+ T cells (Ly2 TsF) can suppress the in vitro response to sheep erythrocytes (SRBC) of spleen cells depleted of Ly2+ T cells. This suppressor factor expresses biological activity only when the acceptor cells share major histocompatibility complex (MHC)-linked polymorphic genes with the cells that made the Ly2 TsF. Removal of Ly1 I-J+ cells from the assay culture abrogates the ability of Ly2 TsF to suppress these cultures, but we can replace the need for the I-J+ cells in the assay culture with an I-J+ soluble factor derived from them. We investigated the cellular interactions involved in the activation of I-J+ cells by Ly2 TsF in vitro. We have been able to induce the production of an I-J+ molecule needed for Ly2 TsF activity in a 48-h intermediate culture of B cell-depleted Ly1 spleen cells, Ly2 TsF, and antigen. This molecule not only fails to bind antigen, but is also antigen nonspecific in that it can be induced by Ly2 TsF of irrelevant specificities. In order to replace the activity of the Ly1 I-J+ cell in the assay culture, the cell induced by Ly2 TsF to produce the I-J+ molecule in vitro must share genetic polymorphisms linked to the MHC with the Ly2 TsF, and genetic polymorphisms linked to the Igh-V gene complex with the target cell. In order for Ly2 TsF to induce cells of the primary culture to produce the I-J+ molecule, Ly2 TsF must share genetic polymorphisms linked to the IE region of the MHC with the Ly1 I-J+ cell producing the I-J+ molecule. These results indicate that the suppressive mechanism of Ly2 TsF involves the interaction with an Ly1 I-J+ molecule. This I-J+ molecule serves to focus the antigen-specific suppressor molecule on the target cell. The recognition event of this suppressive complex on the surface of the acceptor cell is controlled by Igh-V-linked genes restricted by the I-J+ molecule of the suppressor complex. This suppressor interaction is confined to the suppressor effector phase of the suppressor circuit since the I-J+ molecules needed for by Ly2 TsF activity do not substitute for the I-J+ molecules needed for the activity of Ly1 TsiF , a T cell factor that initiates the suppressor cell circuit.(ABSTRACT TRUNCATED AT 400 WORDS)

Human Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes home preferentially to and induce selective regressions of autologous EBV-induced B cell lymphoproliferations in xenografted C.B-17 scid/scid mice.

C.B-17 scid/scid (severe combined immunodeficiency [SCID]) mice inoculated with peripheral blood lymphocytes from Epstein-Barr virus (EBV)-seropositive donors, or with EBV-transformed lymphoblastoid B cell lines (EBV-LCL), develop lethal human EBV+ B cell lymphoproliferative disorders (EBV-LPD) with characteristics similar to those arising in immunodeficient patients. Using this model, we examined the capacity of human effector cells to control human EBV-LPD. SCID mice received rabbit anti-asialo GM1 antiserum to abrogate endogenous natural killer-cell function. Preliminary experiments showed that adoptive transfer of peripheral blood mononuclear cells (PBMC), purified T cells, interleukin (IL) 2-activated PBMC or anti-CD3-activated T cells derived from EBV-seropositive donors did not result in improved survival of treated mice (in vivo effector/target ratio 2:1 to 1:1). In contrast, EBV-specific cytotoxic T lymphocytes (CTL), derived from EBV-seropositive donors and expanded in vitro, exhibited strong EBV-specific and HLA-restricted activity both in vitro and in vivo. SCID mice inoculated intraperitoneally with autologous but not with HLA-mismatched EBV-LCL had significantly improved survival relative to untreated mice after inoculation of EBV-specific CTL either intraperitoneally (P<0.001) or intravenously (P<0.001) (in vivo effector/target ratio 1:1). SCID mice bearing large subcutaneous EBV+ tumors and treated intravenously with 10(7) EBV-specific CTL achieved complete tumor regression. Both CTL- and CTL-plus-IL-2-treated mice survived significantly longer than untreated animals or animals treated with IL-2 alone (P = 0.0004 and P<0.02, respectively). SCID mice bearing two subcutaneous EBV+ tumors, one autologous and the other HLA mismatched to the EBV-specific CTL donor, had regression of only the autologous tumor after intravenous infusion of 10(7) EBV-specific CTL. Moreover, we could demonstrate preferential homing of PKH26-labeled EBV-specific CTL to autologous but not to HLA-mismatched EBV+ tumors as early as 24 h after intravenous adoptive transfer. Immunophenotypic analyses also demonstrated preferential infiltration of T cells into the autologous EBV+ tumor in SCID mice bearing both the autologous and either fully HLA-mismatched or genotypically related haplotype-sharing EBV+ tumors. The human T cells infiltrating EBV+ tumors were CD3+ and, predominantly, CD8+CD4-. Our results indicate that EBV-specific CTL preferentially localize to and infiltrate EBV+ tumors bearing the appropriate HLA antigens and thereafter induce targeted regressions of disease.

The t(2;3)(q21;q27) translocation in non-Hodgkin's lymphoma displays BCL6 mutations in the 5' regulatory region and chromosomal breakpoints distant from the gene.

The BCL6 gene, mapped at the chromosomal band 3q27, encodes a POZ/Zinc finger transcription repressor protein. It is frequently activated in Non-Hodgkin's lymphomas (NHL) by translocations with breakpoints clustering in the 5' major breakpoint region (MBR) as well as by mutations in the same region. The translocations lead to BCL6 activation by substitution of promoters of rearranging genes derived from the reciprocal chromosomal partners such as IG. We report the molecular genetic analysis of a novel t(2;3)(q21;q27) translocation subset in NHL comprising three cases without apparent BCL6 involvement in the translocation. Southern blot analysis of tumor DNAs utilizing BCL6 MBR probes revealed no rearrangement in two cases. Two rearranged bands in the third case resulted from a deletion in one allele and a mutation in the other allele. Southern blot analysis of DNA from one of the two tumors without BCL6 rearrangement, using a probe derived from the recently identified alternative breakpoint region (ABR), showed a rearrangement. The ABR is located 200-270 kb telomeric to MBR. Mutations were identified in the previously reported hypermutable region of BCL6 in all three tumors. In one, the mutant allele alone was found to be expressed by RT-PCR analysis of RNA. These results demonstrate the presence of 3q27 translocation breakpoints at a distance from BCL6 suggesting distant breaks that deregulate the gene or involvement of other genes that may be subject to rearrangement.

Molecular cloning and DNA sequence analysis of cDNA encoding chicken homologue of the Bcl-2 oncoprotein.

We have isolated a 2228 bp cDNA clone encoding a chicken homologue of the human Bcl-2 oncoprotein by low-stringency hybridization screening of a lambda gt10 cDNA library derived from a chicken B-cell lymphoma. DNA sequence analysis of this cDNA revealed an open reading frame predicting a polypeptide of 232 amino acids and an M(r) of 25,839. The predicted protein is highly homologous to the human (73%) and mouse (70%) Bcl-2 proteins, and contains a hydrophobic stretch of amino acids within its carboxyl-end (213-229) consistent with an integral membrane protein. Areas of very high sequence homology shared by all three Bcl-2 proteins at the NH2-terminus (amino acids 1-33) and within the last 150 amino acids of these proteins suggest the presence of at least two evolutionarily conserved domains within the family of Bcl-2 proteins that may be important either for their targeting to mitochondria or their ability to block programmed cell death.

Expression of insulin receptor mRNA and insulin receptor substrate 1 in pancreatic islet beta-cells.

The expression of insulin receptor mRNA was examined in rat pancreatic islet cells by single-cell reverse transcriptase (RT)-polymerase chain reaction (PCR). Single cells from disaggregated islets were individually isolated in a microcapillary pipet, and the beta-cells were identified by amplification of the mRNA for insulin. We found that in single beta-cells, the mRNA for the insulin receptor was also expressed. The fraction of single islet cells expressing both insulin receptor and insulin mRNAs corresponds closely to the fraction of beta-cells in the disaggregated islet cell preparation. These results indicate that normal beta-cells have the potential to express authentic insulin receptors. Immunohistochemical analysis was insufficiently sensitive for assaying insulin receptor protein; however, insulin receptor substrate 1 (IRS-1) was readily immunolocalized in islet beta-cells. Since IRS-1 links several cell surface receptors, including those for insulin and IGF-I, to distal signal transduction pathways, our observations indicate that hormonal regulation of islet beta-cells potentially involves the same signal transduction pathway that mediates insulin and growth factor signaling in peripheral insulin target tissue cell types.

Blastic natural killer cell leukemia/lymphoma: a clinicopathologic study.

The classification of natural killer (NK)-cell and NK-like T-cell malignancies has undergone significant evolution in recent years. Although examples of NK-cell tumors resembling acute leukemia have been described anecdotally as blastic, blastoid, or monomorphic NK-cell leukemia/lymphoma (NKL/L), the clinical and pathologic features of these tumors have not been systematically defined. We report four patients with blastic NKL/L and describe the clinical, pathologic, and immunophenotypic findings in these cases. All patients were elderly (58-82 years) and presented with cutaneous plaques. Two patients also had adenopathy, and three patients had marrow involvement at presentation. Biopsy of cutaneous lesions showed atypical superficial and deep dermal lymphoid infiltrates. Involved lymph nodes were architecturally effaced by an interfollicular infiltrate with blastic cytologic features. In Wright-Giemsa-stained blood or marrow smears, tumor cells had finely distributed nuclear chromatin, many with nucleoli, and variable amounts of cytoplasm. In contrast to many NK and NK-like T-cell disorders, azurophilic cytoplasmic granules were absent or inconspicuous. The tumor cells were immunophenotypically distinctive. They expressed intermediate density CD45, as is characteristic of blasts; in addition, the cells were positive for HLA-DR, CD2, CD4, and the NK-associated antigen CD56. Surface CD3, cytoplasmic CD3, and CD5 were negative in all cases tested, whereas CD7 was expressed in two cases. In formalin-fixed tissue, tumor cells marked with antibodies to CD43, but not with other T- or B-lineage-related antibodies. All three cases studied for Epstein-Barr viral RNA by in situ hybridization were negative. Although treatments varied, all three patients with clinical follow-up died within months of the diagnosis. The clinical course in two patients culminated in an overtly leukemic phase. These findings suggest that blastic NKL/L represents a distinct clinicopathologic entity, characterized by cutaneous, nodal, and marrow involvement by blastic cells with immunophenotypic characteristics of true NK cells. The disease afflicts elderly patients, pursues an aggressive course, and may culminate in overt leukemia.

Nonsyntenic amplification of MYC with CDK4 and MDM2 in a malignant mixed tumor of salivary gland.

Karyotypic analysis of a metastatic malignant mixed tumor of the salivary gland revealed the presence of double minute chromosomes (dmin), indicative of gene amplification. Comparative genomic hybridization analysis of DNA extracted from the primary and a renal metastasis indicated overt amplification of DNA sequences derived from 8q23-24 and 12q13-15 regions. Subsequent Southern blot analysis of tumor DNA from the metastasis with the use of probes previously mapped to those regions indicated amplification of MYC at 8q23-24 and CDK4 and MDM2 at 12q13-15. Fluorescence in situ hybridization of differentially labeled MYC and MDM2 genes hybridized to tumor metaphase chromosomes revealed an independent nonsyntenic amplification of MYC and MDM2 on dmin in this tumor.

BCL6 gene rearrangement and other cytogenetic abnormalities in diffuse large cell lymphoma.

Evidence for rearrangement of the BCL6 gene at 3q27 has been documented in 20-30% diffuse lymphomas with a large cell component (DLLC), and was found to be of prognostic significance at the time of diagnosis. To incorporate these observations into current cytogenetic and clinical prognostic models, 76 cases of DLLC with known BCL6 status were analyzed. Cytogenetic indicators of progression, including trisomy 7, trisomy 12, del(6)(q21q25), and structural alterations of 17p were less frequent in BCL6 rearranged DLLC compared to BCL6 germline tumors. Despite a 93% overall survival at median follow-up of 30 months, a trend for continued relapse resulted in a projected freedom from progression for the BCL6 rearranged cohort of 66% at 4 years, compared to 39% for the BCL6 germline cohort. Six cases among the BCL6 rearranged group lacked additional cytogenetic indicators of progression and remained free of disease at follow-up in excess of 7 years, whereas BCL6 rearranged cases with increasing numbers of cytogenetic aberrations showed decreased intervals free from progression of disease. These results suggest that BCL6 rearrangement should be combined with other known clinical and cytogenetic indicators in prognostic analyses of patients with DLLC.

Del (7)(q32) is associated with a subset of small lymphocytic lymphoma with plasmacytoid features.

Deletions of the long arm of chromosome 7, previously documented in myelodysplasias and myeloid leukemias, have also been noted in lymphoid malignancies. Of 558 karyotypically abnormal specimens of non-Hodgkin's lymphoma (NHL) serially ascertained over an 8-year period, del(7q) was identified in 24 cases, 10 of which were of the small lymphocytic (sm lym) subtype. Del(7q) was the third most common karyotypic abnormality among the cohort of 61 sm lym cases in this ascertainment. Mapping of the deletions identified a region of common deletion affecting 7q32, which was the sole karyotypic abnormality in 2 cases. Eight of the ten sm lym cases were characterized by plasmacytoid features in histologic sections of lymphoma tumors or circulating cells in the peripheral blood. The del(7)(q32) was accompanied by 14q32-associated translocations in 11 of the 14 cases with histologies other than sm lym, compared with 2 of the sm lym cases. Extranodal involvement was more frequent in the del(7)(q32) sm lym NHLs, although median survival was typical of other low-grade lymphomas. These results suggest that loss or inactivation of a putative tumor-supressor gene at 7q32 may play a role the progression of lymphomas as well as constitute an early event in the pathogenesis of lymphoplasmacytoid tumors.

Non-Hodgkin lymphoma of the breast: imaging characteristics and correlation with histopathologic findings.

PURPOSE: To determine the imaging characteristics of non-Hodgkin lymphoma (NHL) of the breast and correlate these findings with histopathologic subtype. MATERIALS AND METHODS: The authors retrospectively reviewed records in 32 cases of histologically proved NHL in 29 women. Mammography was performed before biopsy in all cases, and breast sonography was performed before biopsy in eight. RESULTS: Mammary NHL was primary in 21 of the 32 cases (66%) and secondary in 11 (34%). Mammography revealed a solitary, uncalcified mass in 22 cases (69%), multiple masses in three (9%), and diffuse increased opacity with skin thickening in three (9%). Four cases (13%) had normal findings. Seven of the eight sonograms revealed masses, which were solitary in five and multiple in two. Histologic examination showed diffuse NHL in 26 cases (81%) and follicular NHL in six (19%). No mammographic or sonographic features were identified that helped distinguish primary from secondary disease or follicular from diffuse NHL. CONCLUSION: Although the imaging characteristics may suggest the possibility of breast NHL, none of the findings are pathognomonic. The imaging pattern of mammary NHL is unrelated to its histopathologic subtype.

Diffuse large cell lymphomas exhibit frequent deletions in 9p21-22 and 9q31-34 regions.

We have previously identified deletions of 9p and 9q in a cytogenetic analysis of a large series of non-Hodgkin's lymphomas (NHLs), which suggested loss of candidate tumor suppressor genes (TSGs). In order to define these deletions at the molecular level, we performed an LOH analysis of a panel of paired normal and tumor DNAs comprising 13 cases of diffuse lymphoma with a large cell component (DLLC) and 18 cases of Burkitt's lymphoma (BL). The loci tested comprised eight polymorphic probes mapped to 9p (D9S33, D9S25, IFNB, IFNA, IFNW, D9S126, D9S3, and D9S19) and seven polymorphic probes mapped to 9q (D9S29, ASS, AKI, ABL, D9S10, D9S7, and D9S14). In this analysis, among cases informative for all loci in each subset, 5/13 (38%) DLLC and 4/18 (22%) BL showed LOH at 9p loci, whereas 5/13 (38%) DLLC and 3/18 (16%) BL showed LOH at 9q loci. Among the 9p loci partial homozygous or heterozygous losses were observed in 20-50% of informative cases of DLLC at D9S25, IFNB, IFNA, IFNW, D9S126, and D9S3, whereas in BL, losses at these loci ranged from 0% to 11%. Among the 9q loci, heterozygous losses were observed in > 20% of informative cases of DLLC at D9S7 (23%) and D9S29 (27%), whereas no losses were seen at these two loci in BL. These data demonstrate a high level of molecular deletion in DLLC, but not in BL, suggesting that loss of one or more TSGs on chromosome 9 plays an important role in DLLC development.

Absence of t(14;18) major and minor breakpoints and of Bcl-2 protein overproduction in Reed-Sternberg cells of Hodgkin's disease.

The t(14;18) translocation, found in most human follicular non-Hodgkin's lymphomas (NHLs), juxtaposes the Bcl-2 oncogene at 18q21 with the immunoglobulin heavy chain locus at 14q32. As a result, the Bcl-2 protein is markedly overproduced. Most of the breakpoints on chromosome 18 cluster at one of two sites, the major breakpoint region (mbr) and the minor cluster region (mcr). Recently, others used the polymerase chain reaction (PCR) to detect the t(14;18) mbr in 32% of specimens diagnosed as Hodgkin's disease (HD). In an attempt to confirm and extend those observations the authors used PCR to assay for both the mbr and mcr in HD specimens diagnosed at their institution and examined the specimens for Bcl-2 overproduction. The authors subjected the DNAs from 28 well-characterized HD tumors of 26 patients to PCR analyses using primers specific for the t(14;18) mbr and mcr breakpoints. Based on various PCR controls, the authors ascertained that 26 of the 28 specimens contained amplifiable template DNA. Southern blotting of the amplification products showed that none of the 26 HD DNAs had detectable t(14;18) mbr or mcr breakpoints. By admixing small amounts of t(14;18)-bearing NHL DNA with HD DNA samples, the authors directly demonstrated that the sensitivity of the PCR assays was adequate for the molecular detection of t(14;18)-bearing cells at a frequency comparable to that of Reed-Sternberg cells and their variants in HD. Immunohistochemical studies employing a highly specific anti-Bcl-2 antiserum under conditions optimized to detect t(14;18)-mediated overexpression of the Bcl-2 gene showed that the Reed-Sternberg cells and variants in all 19 HD tumors examined were negative for Bcl-2 immunostaining. In conclusion, the PCR and immunohistochemical data provided evidence that the t(14;18) translocation was not involved in the pathogenesis of the HD cases.

Frequent incidence of somatic mutations in translocated BCL2 oncogenes of non-Hodgkin's lymphomas.

The majority of non-Hodgkin's B-cell lymphomas contain a t(14;18) translocation that places the bc12 gene into juxtaposition with the transcriptically active Ig heavy-chain locus, thus deregulating the expression of this proto-oncogene. The bc12 gene product is a membrane-associated mitochondrial protein that regulates cell survival through unknown mechanisms. Although overproduction of the normal protein appears sufficient for conferring a selective growth or survival advantage to B cells, point mutations that alter the coding region of translocated bc12 genes have been described previously by others in a lymphoma cell line. However, it is not known whether somatic mutations that alter BCL2 proteins occur in vivo or whether they result from chemotherapy or arise through other mechanisms. For these reasons, we obtained DNA from the t(14;18)-containing tumors of five patients who had not undergone treatment for their disease, and used a polymerase chain reaction (PCR)-mismatch technique for rapid identification of point mutations in a portion of the bc12 open reading frame (ORF) corresponding to the first 131 aminoacids (aa) of the 239 aa p26 BCL2 protein. DNAs from two t(14;18)-containing cell lines were also analyzed. Point mutations in this region of the bc12 gene ORF were detected in three of five patients' tumors and in both cell lines. PCR-mismatch analysis of bc12 in cell lines and non-Hodgkin's lymphoma cases that lacked the t(14;18) translocation was negative, thus establishing the specificity of these results. DNA sequencing determined that these mutations are predicted to produce aa substitutions in the BCL2 proteins of two of the primary tumors and one of the cell lines. Interestingly, two of the patients contained an identical C----T transition that resulted in a nonconservative aa substitution (proline----serine) at position 59 of the BCL2 protein. Further analysis excluded the possibility that these mutations represented hereditary polymorphisms or PCR artifacts. A cluster of four point mutations within the translocation + bc12 allele of one patient had hallmarks of the somatic hypermutation mechanism that is associated with Ig genes and that contributes to antibody diversity. Because of the region of the bcl2 gene analyzed in these t(14;18) translocations is located nearly 300 kbp from the Ig heavy-chain locus, our data suggest that the Ig gene somatic hypermutation mechanism can act over extreme distances of DNA. It remains to be established whether these somatic mutations that alter BCL2 proteins influence the pathobiology of nonHodgkin's lymphomas.

Heterologous promoters fused to BCL6 by chromosomal translocations affecting band 3q27 cause its deregulated expression during B-cell differentiation.

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.

p53 mutations are associated with histologic transformation of follicular lymphoma.

The majority of low-grade non-Hodgkin's lymphomas (NHL) undergo clinical progression toward intermediate- and high-grade lymphomas. This progression is often associated with histologic transformation from follicular to diffuse-type NHL. The pathogenetic mechanisms underlying this evolution are presently unknown. In this study, we have analyzed the role in NHL progression of relevant genetic lesions affecting proto-oncogenes and tumor suppressor genes. Sequential biopsies from 21 patients with clinical progression with (5 cases) or without (16 cases) evidence of histologic transformation were analyzed for karyotypic changes, c-myc rearrangements and deletions affecting 6q27 by Southern blot analysis, and p53 mutations by single-strand conformation polymorphism (SSCP) analysis coupled with direct sequencing of polymerase chain reaction-amplified products. No novel cytogenetic aberration was detected in association with progression, and all samples analyzed displayed a normal c-myc gene. Mutations of the p53 gene were detected in 4 of 5 cases displaying histologic transformation from follicular to diffuse-type NHL and in none of the 16 cases displaying clinical progression in the absence of histologic transformation. In 1 of these positive cases, the same mutation was also present in the pretransformation biopsy, correlating with the presence of diffuse-type areas within a predominant follicular pattern. In 1 of these cases, a deletion of 6q27 was also detected in the posttransformation biopsy along with a p53 mutation. These findings indicate that p53 mutations are associated with and may be responsible for histologic transformation of follicular lymphoma.

Clinical and morphologic features of B-cell small lymphocytic lymphoma with del(6)(q21q23).

Deletions of the long arm of chromosome 6 have been described in acute and chronic lymphocytic leukemia (ALL and CLL) and prolymphocytic leukemia (PLL), and have been associated with t(14;18)(q32;q21) in non-Hodgkin's lymphoma (NHL). Of 55 cases of small lymphocytic (sm lym) NHL, deletions of 6(q21q23) were the most common recurring cytogenetic abnormality. Among 14 sm lym NHL with del(6)(q21q23), this abnormality occurred as a solitary change in 3 cases. Each of these 3 cases, and 5 additional cases with del(6q) and other abnormalities, showed atypical larger forms with the morphologic appearance of prolymphocytes or paraimmunoblasts in the peripheral blood. In comparison, of the 11 cases without del(6q) and circulating abnormal cells, prolymphocytoid forms were observed in 4 cases (P < .001). Of the 31 sm lym without del(6q), trisomies of chromosomes 3, 12, or 18, or t(11;14)(q13;q32) occurred in greater than 10% of cases. Proliferation centers or infiltration by larger forms were observed in similar proportions of tissue sections derived from sm lym NHL with or without del(6q). The presence of the larger forms in the peripheral blood did not have an adverse prognostic impact on the survival of the del(6q) cohort, who experienced a median survival in excess of 6 years. All 14 cases of del(6q) sm lym NHL were characterized by a mature B-cell phenotype. Expression of CD11c, a feature of a CLL/PLL variant previously described, was not detected in 9 cases analyzed. In 5 cases of del(6q) sm lym NHL, no circulating abnormal lymphocytes were noted. Twelve cases presented with, or developed, clinical splenomegaly. These results suggest that deletion of a gene or genes at 6q21-23 is associated with the pathogenesis of a subset of B-cell sm lym NHL that may display larger prolymphocytoid cells in the peripheral blood, but that follows a clinical course typical of other well-differentiated lymphocytic neoplasms.

Involvement of BCL6 in chromosomal aberrations affecting band 3q27 in B-cell non-Hodgkin lymphoma.

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.

p53 overexpression as a marker of poor prognosis in mantle cell lymphomas with t(11;14)(q13;q32).

The t(11;14)(q13;q32) translocation, which juxtaposes the BCL1 oncogene with the Ig heavy chain locus, has been associated with an uncommon subtype of non-Hodgkin's lymphoma (NHL) termed mantle cell lymphoma (MCL). To date, no molecular marker that serves as an indicator of tumor progression or clinical prognosis has been described for NHLs with this translocation. We examined a panel of NHLs with t(11;14) for overexpression of p53 and correlated the results with single-strand conformation polymorphism (SSCP) analysis, karyotypic features, and clinical course. NHLs with t(11;14) were identified from 30 patients. The diagnosis was MCL for 23 of 30, small lymphocytic lymphoma for 4 of 30, and diffuse large-cell lymphoma for 3 of 30 cases. The results of immunohistochemistry analysis using a monoclonal anti-p53 antibody on paraffin-embedded specimens were compared with the SSCP data, the tumor karyotypes, and clinical course of each patient. DNA sequencing of exons was performed on cases that showed conformational changes by SSCP analysis. NHLs from 5 of 23 patients with MCL were positive for p53 overexpression. Deletions of chromosome 17p were identified in 2 of 30 cases, both of which were MCLs showing p53 overexpression. Two of the five MCLs with p53 overexpression showed evidence for TP53 mutations. None of the 18 MCLs negative for p53 overexpression showed conformational changes by SSCP. For these 18 patients with MCLs that did not overexpress p53, the median survival was 63 months, compared with 12 months for the 5 patients with MCLs positive for p53 overexpression (P < .001). These results suggest that p53 overexpression in MCL with t(11;14)(q13;q32) may serve as a marker of poor prognosis.

Inhibition of thymocyte apoptosis and negative antigenic selection in bcl-2 transgenic mice.

The bcl-2 gene, which is overexpressed in human follicular B-cell lymphomas, has been found to extend cellular lifespan through inhibition of apoptosis, or programmed cell death. However, the physiological role of the Bcl-2 protein in lymphocyte development is unclear. We have established a transgenic mouse line that expresses high levels of the Bcl-2 protein in both cortical and medullary thymocytes, disrupting the normal pattern of expression of this gene. We found that in these mice, immature thymocytes became resistant to apoptosis mediated by corticosteroids and calcium ionophores. Untreated thymocytes also exhibited a survival advantage in suspension cultures compared with controls. In addition, overexpression of bcl-2 enabled a proportion of thymocytes and peripheral T cells to escape the process of clonal deletion, which normally eliminates self-reactive T cells during thymocyte maturation. These findings implicate the Bcl-2 protein in regulating the lifespan of maturing thymocytes and in the antigenic-selection process.

Cyclin D1 and retinoblastoma protein expression in Kaposi's sarcoma.

Cyclins are implicated in the induction and control of the cell cycle. Cyclin D1 regulates G1-phase progression by phosphorylation of the retinoblastoma protein (pRb). The Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV) contains and transcribes an open reading frame with sequence similarities to cellular D-type cyclins. The KSHV-cyclin protein is associated with kinase activity capable of phosphorylating pRb in vitro. Here, we study for the first time the endogenous cyclin D1 and Rb protein expression in Kaposi's sarcoma (KS) tissue. Twenty-four consecutive biopsies of AIDS-related (n=21) and classical (n=3) KS were studied by immunohistochemistry with monoclonal antibodies against cyclin D1 and pRb. We detected cyclin D1 in 1 of 13 patch/plaque stage, in 4 of 5 nodular stage and in 3 of 6 visceral KS lesions. By Western blot analysis, this cellular cyclin D1 monoclonal antibody did not cross-react with the purified KSHV-cyclin protein. The pRb was consistently detected in 24 of 24 KS lesions. In summary, early KS lesions rarely have detectable expression of endogenous cyclin D1. Advanced and disseminated KS lesions tend to have overexpression of endogenous cyclin D1. Therefore, cellular cyclin D1 expression appears to correlate with tumor progression in KS. The endogenous cyclin D1 is antigenically distinct from the KSHV-cyclin homolog. The pRb, which may serve as a substrate for KSHV-cyclin, is found in all KS lesions examined.