Ki-67 is a strong predictor of central nervous system relapse in patients with mantle cell lymphoma (MCL).

BACKGROUND: Central nervous system (CNS) relapse is an uncommon but challenging complication in patients with mantle cell lymphoma (MCL). Survival after CNS relapse is extremely poor. Identification of high-risk populations is therefore critical in determining patients who might be candidates for a prophylactic approach. PATIENTS AND METHODS: A total of 608 patients (median age, 67 years; range 22-92) with MCL newly diagnosed between 1994 and 2012 were evaluated. Pretreatment characteristics and treatment regimens were evaluated for their association with CNS relapse by competing risk regression analysis. RESULTS: None of the patients received intrathecal prophylaxis. Overall, 33 patients (5.4%) experienced CNS relapse during a median follow-up of 42.7 months. Median time from diagnosis to CNS relapse was 20.3 months (range: 2.2-141.3 months). Three-year cumulative incidence of CNS relapse was 5.6% [95% confidence interval (95% CI) 3.7% to 8.0%]. Univariate analysis revealed several risk factors including blastoid variant, leukemic presentation, high-risk MCL International Prognostic Index and high Ki-67 (proliferation marker). Multivariate analyses revealed that Ki-67 ≥ 30 was the only significant risk factor for CNS relapse (hazard ratio: 6.0, 95% CI 1.9-19.4, P = 0.003). Two-year cumulative incidence of CNS relapse in patients with Ki-67 ≥ 30 was 25.4% (95% CI 13.5-39.1), while that in the patients with Ki-67 < 30 was 1.6% (95% CI 0.4-4.2). None of the treatment modalities, including rituximab, high-dose cytarabine, high-dose methotrexate or consolidative autologous stem-cell transplant, were associated with a lower incidence of CNS relapse. Survival after CNS relapse was poor, with median survival time of 8.3 months. There was no significant difference in the survival by the site of CNS involvement.

T-cell large granular lymphocytic leukemia occurring after autologous peripheral blood stem cell transplantation.

A 61-year-old man with angioimmunoblastic lymphoma in first complete remission underwent autologous peripheral blood stem cell transplantation. At 1 month post transplant, asymptomatic large granular lymphocytosis developed. The surface marker profile of the cells was CD3+CD8+CD56-CD57+. The disease course was chronic and indolent. The patient remains in complete remission from angioimmunoblastic lymphoma more than 6 months post transplant with persistent large granular lymphocytosis (lymphocyte count, 5-15 x 10(9)/l). Although post transplantation T-cell lymphoproliferative disorders have mostly occurred in allogeneic transplantation recipients and presented as aggressive lymphomas/leukemias, we suggest that chronic indolent T-cell large granular lymphocytic leukemia can occur after autologous stem cell transplantation.

Low frequency of BCL-2/J(H) translocation in peripheral blood lymphocytes of healthy Japanese individuals.

The incidence of follicular lymphoma differs significantly between white and Japanese individuals. Translocation between the BCL-2 and immunoglobulin heavy chain genes is detected in 85% to 90% of all follicular lymphomas in whites. Recently, BCL-2/J(H) translocation was detected in peripheral blood lymphocytes from more than 50% of healthy white individuals. To clarify the reason for the difference in incidence of follicular lymphoma between whites and Japanese, the frequency of BCL-2/J(H) translocation in peripheral blood lymphocytes of healthy Japanese individuals was compared with that of German individuals. The prevalence of BCL-2/J(H) translocation in Japanese adults appeared to be significantly lower than that in German adults. The present data suggest that the low frequency of BCL-2/J(H) translocation in the Japanese general population may be one of the major reasons for the difference in incidence of follicular lymphoma between whites and Japanese.

BAL is a novel risk-related gene in diffuse large B-cell lymphomas that enhances cellular migration.

Clinical risk factor models such as the International Prognostic Index are used to identify diffuse large B-cell lymphoma (DLB-CL) patients with different risks of death from their diseases. To elucidate the molecular bases for these observed clinical differences in outcome, differential display was used to identify a novel gene, termed BAL (B-aggressive lymphoma), which is expressed at significantly higher levels in fatal high-risk DLB-CLs than in cured low-risk tumors. The major BAL complementary DNA encodes a previously uncharacterized 88-kd nuclear protein with a duplicated N-terminal domain homologous to the nonhistone portion of histone-macroH2A and a C-terminal alpha-helical region with 2 short coiled-coil domains. Of note, the BAL N-terminus and secondary structure resemble those of a recently identified human protein, KIAA1268. In addition, both BAL and KIAA1268 map to chromosome 3q21, further suggesting that these genes belong to a newly identified family. BAL is expressed at increased levels in DLB-CL cell lines with an activated peripheral B cell, rather than a germinal center B cell, phenotype. This observation and the characteristic dissemination of high risk DLB-CLs prompted studies regarding the role of BAL in B-cell migration. In classical transwell assays, stable BAL-overexpressing B-cell lymphoma transfectants had significantly higher rates of migration than vector-only transfectants, indicating that the risk-related BAL gene promotes malignant B-cell migration. (Blood. 2000;96:4328-4334)

Stromal cells in lymph nodes attract B-lymphoma cells via production of stromal cell-derived factor-1.

Stromal cell-derived factor-1 (SDF-1) is a chemokine produced by bone marrow stromal cells which plays an important role in B-lymphopoiesis and the homing of hematopoietic stem cells to the bone marrow. In the present study, we investigated the role of SDF-1 and its receptor, CXCR4, in the chemotactic interaction between non-Hodgkin B-lymphoma cells and lymph node stromal cells. SDF-1 mRNA was abundantly expressed in stromal cells isolated from the lymph nodes of patients with malignant lymphoma. All B-lymphoma cells freshly isolated from these patients and most laboratory B-lymphoma cell lines, including follicular, diffuse large, and Burkitt's lymphoma cells, expressed surface CXCR4 and migrated in the presence of recombinant human SDF-1alpha. Chemotaxis assays revealed that CXCR4-positive (but not CXCR4-negative) B-lymphoma cells migrated towards lymph node stromal cells, and this migration was almost completely inhibited by the addition of anti-CXCR4 monoclonal antibody to the lymphoma cells or of anti-SDF-1 neutralizing antibody to the culture supernatant of the stromal cells. Down-regulation of surface CXCR4 was detected in B-lymphoma cells which migrated towards the stromal cells but not in those which showed no migratory response. In addition, contact between the lymphoma cells and the stromal cells resulted in down-regulation of surface CXCR4 on the lymphoma cells. These data strongly suggest that SDF-1/CXCR4 is the main chemokine system involved in the chemotactic interaction between B-lymphoma cells and lymph node stromal cells.

PTPROt: an alternatively spliced and developmentally regulated B-lymphoid phosphatase that promotes G0/G1 arrest.

Protein tyrosine phosphatases (PTP) regulate the proliferation, differentiation, and viability of lymphocytes by modulating their signaling pathways. By using the differential display assay, we have cloned a putative receptor-type PTP, which is predominantly expressed in B-lymphoid tissues (lymph nodes and spleen). This PTP, termed PTPROt (truncated), is a tissue-specific alternatively-spliced form of a human epithelial PTP, PTPRO (PTPU2/GLEPP1). Whereas the epithelial PTPRO includes an approximately 800-amino acid extracellular domain, the major (3 kb) PTPROt cDNA predicts a unique 5' untranslated region and truncated (8 amino acids) extracellular domain with a conserved transmembrane region and single catalytic domain. PTPROt cDNAs encode functional approximately 47-kD and approximately 43-kD PTPs, which are most abundant in normal naive quiescent B cells and decreased or absent in germinal center B cells and germinal center-derived diffuse large B-cell lymphomas. Because PTPROt was predominantly expressed in naive quiescent B cells, the enzyme's effects on cell-cycle progression were examined. When multiple stable PTPROt sense, antisense, and vector only B-cell transfectants were grown in reduced serum and synchronized with nocodazole, PTPROt sense clones exhibited markedly increased G0/G1 arrest. Taken together, these data implicate PTPROt in the growth control of specific B-cell subpopulations.

Fas-independent cytotoxicity mediated by human CD4+ CTL directed against herpes simplex virus-infected cells.

The present study was undertaken to clarify the mechanisms of cytotoxicity mediated by virus-specific human CD4+ CTLs using the lymphocytes of family members with a Fas gene mutation. CD4+ CTL bulk lines and clones directed against HSV-infected cells were established from lymphocytes of a patient with a homozygous Fas gene mutation and of the patient's mother. HSV-specific CD4+ CTLs generated from lymphocytes of the patient and her mother exerted cytotoxicity against HSV-infected cells from the patient (Fas-/-) and from her mother (Fas+/-) to almost the same degree in an HLA class II-restricted manner. mRNAs for the major mediators of CTL cytotoxicity, Fas ligand, perforin, and granzyme B, were detected in these CD4+ CTLs using the RT-PCR and flow cytometry. The cytotoxicity of the HSV-specific CD4+ CTLs appeared to be Ca2+-dependent and was almost completely inhibited by concanamycin A, a potent inhibitor of the perforin-based cytotoxic pathway. Although the Fas/Fas ligand system has been reported to be the most important mechanism for CD4+ CTL-mediated cytotoxicity in the murine system, the present findings strongly suggest that granule exocytosis, not the Fas/Fas ligand system, is the main pathway for the cytotoxicity mediated by HSV-specific human CD4+ CTLs.

Down-regulation of CXCR4 by human herpesvirus 6 (HHV-6) and HHV-7.

Recent studies have demonstrated that human herpesvirus 6 (HHV-6) and HHV-7 interact with HIV-1 and alter the expression of various surface molecules and functions of T lymphocytes. The present study was undertaken to clarify whether coreceptors for HIV-1, CXCR4 and CCR5, are necessary for HHV-6 and HHV-7 infection. Although CXCR4 and CCR5 appeared not to be the coreceptors for these viruses, marked down-regulation of CXCR4, but not CCR5, was detected in HHV-6 variant A (HHV-6A)-, HHV-6 variant B (HHV-6B)-, and HHV-7-infected cells. Down-regulation of CXCR4 resulted in impairment of chemotaxis and a decreased level of elevation of the intracellular Ca2+ concentration in response to stromal cell-derived factor-1. Northern blot analysis of mRNAs extracted from HHV-6A-, HHV-6B-, and HHV-7-infected CD4+ T lymphocytes demonstrated a markedly decreased level of CXCR4 gene transcription, but the posttranscriptional stability of CXCR4 mRNA was not significantly altered. These data demonstrate that unlike HIV-1, HHV-6 and HHV-7 infections do not require expression of CXCR4 or CCR5, whereas marked down-regulation of CXCR4 is induced by these viruses, suggesting that HHV-6 and HHV-7 infections may render CD4+ T lymphocytes resistant to T lymphocyte-tropic HIV-1 infection.

Fas-independent and nonapoptotic cytotoxicity mediated by a human CD4(+) T-cell clone directed against an acute myelogenous leukemia-associated DEK-CAN fusion peptide.

The mechanism underlying the cytotoxicity mediated by a human CD4(+) cytotoxic T-lymphocyte (CTL) clone directed against a peptide derived from the acute myelogenous leukemia-associated fusion protein, DEK-CAN, was investigated. A DEK-CAN fusion peptide-specific CD4(+) Th0 CTL clone, designated HO-1, was established from the peripheral blood lymphocytes of a healthy individual. HO-1 exerted direct but not "innocent bystander" cytotoxicity within 2 hours. The cytotoxicity mediated by HO-1 was completely Ca2+-dependent. Because HO-1 lysed peptide-loaded Fas-deficient target cells derived from a patient with a homozygous Fas gene mutation, its cytotoxicity appeared to be mediated by a Fas-independent pathway. In addition, its cytotoxicity was only partially inhibited by treatment with concanamycin A and strontium ions, which are inhibitors of the perforin-based cytotoxic pathway. Although membrane-bound type of tumor necrosis factor-alpha (TNF-alpha) was expressed on HO-1, an anti-TNF-alpha antibody had no effect on HO-1-mediated cytotoxicity. HO-1 expressed mRNA for apoptosis-inducing mediators, including perforin, granzyme B, Fas ligand, TNF-alpha, and lymphotoxin; however, no DNA fragmentation was detected in target cells incubated with HO-1 by 5-[125I]Iodo-2'-deoxyuridine release assay and agarose gel electrophoresis of DNA. Although it has been suggested that the Fas/Fas ligand system is the main pathway by which CD4(+) CTL-mediated cytotoxicity is exerted in murine systems, HO-1 produced peptide-specific and HLA-restricted cytotoxicity via a Fas-independent and nonapoptotic pathway. The present study thus describes a novel mechanism of cytotoxicity mediated by CD4(+) CTL.

Latent infection and reactivation of human herpesvirus 6 in two novel myeloid cell lines.

It has been reported that reactivation of human herpesvirus-6 (HHV-6) causes a failure of hematopoiesis. To clarify the mechanisms of bone marrow suppression induced by HHV-6 infection, it is necessary to establish an in vitro model of HHV-6 infection in hematopoietic progenitor cells. We have established two novel Philadelphia chromosome-positive myeloid cell lines, SAS413 and SAS527, which possess different hematologic characteristics and show distinct susceptibility to infection by HHV-6, from a patient with blast crisis of chronic myelogenous leukemia (CML). HHV-6 subgroup A (HHV-6A) showed marked replication in SAS413, forming syncytia and inducing cell lysis in short-term culture. On the other hand, HHV-6A-inoculated SAS527 continued to proliferate without cell lysis and only a few cells showed HHV-6 antigen expression. In contrast to HHV-6A infection, inoculation with HHV-6 subgroup B (HHV-6B) did not induce any cytopathic effect (CPE) or viral antigen expression in either of the cell lines. Although HHV-6B replication was undetectable, the presence of the HHV-6 genome in both cell lines was shown by polymerase chain reaction (PCR) during culture for more than 10 months, suggesting that HHV-6B latently infected SAS413 and SAS527. Phorbol ester treatment of SAS527 latently infected with HHV-6B resulted in reactivation of HHV-6, as shown by the appearance of a CPE, positive reactivity for the HHV-6 antigen, and isolation of infectious HHV-6. These novel cell lines should be useful for studying the mechanisms of HHV-6-induced hematopoietic failure and HHV-6 latency and reactivation, as well as differentiation, of the myeloid cell lineage.

CD4(+) cytotoxic T-cell clones specific for bcr-abl b3a2 fusion peptide augment colony formation by chronic myelogenous leukemia cells in a b3a2-specific and HLA-DR-restricted manner.

Although it is well known that CD8(+) cytotoxic T lymphocytes (CTLs) play an important role in the suppression of cancer cell growth, the significance of CD4(+) CTLs in resistance to cancer is obscure. In an attempt to elucidate the role of CD4(+) CTLs in immunosurveillance of chronic myelogenous leukemia (CML), we examined the immunologic functions of bcr-abl b3a2 fusion peptide-specific CD4(+) CTL clones. Seven CD4(+) T-cell clones that responded to stimulation with b3a2 peptide, but not with b2a2 peptide or physiological counterparts bcr b3b4 and abl 1A-a2 peptides, were established from two healthy individuals. Restriction elements of these clones were HLA-DRB1*0901. These CD4(+) T-cell clones exhibited b3a2 peptide-specific and HLA-DRB1*0901-restricted cytotoxicity and produced interleukin-3 (IL-3), IL-4, IL-10, interferon-gamma, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor in response to bcr-abl peptide stimulation, indicating they were Th0 clones. The numbers of HLA-DRB1*0901-positive b3a2, but not those of b2a2-positive or HLA-DRB1*0901-negative CML cell colonies increased when CML cells were cultured with b3a2-specific CD4(+) CTL clones. These data suggest that bcr-abl-specific CD4(+) CTLs recognize CML cells in an antigen-specific and HLA-DR-restricted manner, and that they do not inhibit, but in fact augment, CML cell growth.

A directly spliced exon 10-containing CD44 variant promotes the metastasis and homotypic aggregation of aggressive non-Hodgkin's lymphoma.

Variants of the CD44 cell-surface adhesion molecule include additional sequences encoded by combinations of exons from the membrane proximal domain (exons 6-14). Preliminary studies suggest that these additional variable membrane proximal sequences may alter the ligand specificity, glycosylation, and biologic function of CD44. In earlier studies, we found that primary extranodal and widely disseminated aggressive non-Hodgkin's lymphomas (NHLs) and normal activated B cells expressed a directly spliced exon 10-containing variant (CD44ex10), whereas normal resting B cells expressed larger exon 10-containing variants (CD44ex10-14 and CD44ex7-14). To obtain additional information regarding the function of exon 10-containing CD44 variants in aggressive NHL, we generated aggressive NHL transfectants that expressed CD44ex10, CD44ex10-14, CD44ex7-14, the standard CD44 isoform (CD44H), or vector alone, and evaluated the local tumorogenicity, aggregation, and metastatic potential of these transfectants. CD44ex10 aggressive NHL transfectants were more likely to cause local tumor formation in nude mice than transfectants expressing the larger exon 10-containing variants, CD44H, or vector alone. In addition, cell suspensions derived from CD44ex10 local tumors exhibited far greater homotypic aggregation than those obtained from other CD44 or vector-only local tumors. In nude mice that received CD44ex10 transfectants, distant metastases were also significantly more likely to develop than in animals that were given either the CD44ex10-14, CD44ex7-14, CD44H, or vector-only transfectants. These data provide the first evidence that the directly spliced exon 10-containing CD44 variant (CD44ex10) has a unique biologic function in aggressive NHL.

Deleted HTLV provirus in peripheral blood cells of a patient with T-cell prolymphocytic leukaemia.

We describe the first case of T-cell prolymphocytic leukaemia (T-PLL) in which the peripheral blood cells contained a human T-lymphotropic virus (HTLV) related tax sequence. Serum screening tests for anti-HTLV-I/II antibodies were negative. Polymerase chain reaction disclosed the presence of an HTLV-I tax sequence in the peripheral blood. Other sets of oligonucleotide primers for HTLV-I gag, pol, env and the long terminal repeat regions and for the HTLV-II pol region were negative in the DNA of the cells. Although patients with T-PLL have been reported to be seronegative for HTLV-I, our findings point to the possibility that HTLV-I infection might be involved in the aetiology of at least some cases of T-PLL and that there may be alternative mechanisms involved in HTLV-associated leukaemogenesis.

Two distinct mechanisms of cytotoxicity mediated by herpes simplex virus-specific CD4+ human cytotoxic T cell clones.

The present study was undertaken to elucidate the mechanisms responsible for the cytotoxicity of herpes simplex virus (HSV)-specific CD4+ human cytotoxic T lymphocyte (CTL) clones, focusing on perforin and membrane-bound lymphotoxin (LT) (tumor necrosis factor-beta). Two HSV-specific CD4+ CTL clones, which expressed both perforin and membrane-bound LT, exerted HSV-specific cytotoxicity and cytotoxicity against LT-sensitive L929 cells. These CD4+ CTL clones lysed HSV-infected cells directly in an HLA class II-restricted manner and did not exhibit "bystander killing." The culture supernatants of these clones stimulated with HSV antigen showed no cytotoxicity against HSV-infected cells or L929 cells, suggesting that adhesion to target cells is essential to their antigen-specific and antigen-nonspecific cytotoxicities. The cytotoxicities of these clones against HSV-infected autologous cells were inhibited by an anti-CD3 monoclonal antibody but not by an anti-LT antibody. Conversely, their cytotoxicities against L929 cells appeared to be partially inhibited by the anti-LT antibody but not by the anti-CD3 monoclonal antibody. Furthermore, target cell DNA fragmentation induced by these CD4+ CTL clones was apparently observed in L929 cells but only faintly detected in HSV-infected autologous cells. L929 cell DNA fragmentation was also inhibited by adding the anti-LT antibody to CD4+ CTL cultures. These data suggest that some CD4+ CTL possess at least two cytolytic mediators, i.e., perforin and membrane-bound LT simultaneously, and can exert both antigen-specific cytotoxicity via two distinct mechanisms, necrosis and apoptosis.

CD4 down-modulation by ganglioside and phorbol ester inhibits human herpesvirus 7 infection.

Recently, data demonstrating that CD4 is an essential component of the receptor for human herpesvirus 7 (HHV-7) as well as for human immunodeficiency virus have been accumulating. Since gangliosides and phorbol esters are known to induce selective down-modulation of cell surface CD4 expression, it might be expected that treatment with these agents would interfere with HHV-7 infection of CD4+ T cells. The present study, undertaken to verify this possibility, demonstrated that addition of monosialoganglioside-GM1 or 12-O-tetradecanoylphorbol 13-acetate effectively induced disappearance of CD4 from the cell surface and also reduced HHV-7 infectivity, as judged by the CPE on virus-infected cells and studies of indirect immunofluorescence, TCID50 and semi-quantitative PCR of the HHV-7 genome. Taken together with previous studies, the present data strongly suggest that the CD4 molecule is a critical component of the receptor for HHV-7.

Distinct effects of human herpesvirus 6 and human herpesvirus 7 on surface molecule expression and function of CD4+ T cells.

This study was undertaken to investigate the effects of newly isolated T lymphotropic viruses, human herpesvirus (HHV)-6 and HHV-7, on CD4+ T cells. We first examined changes in surface molecule expression on CD4+ T cells after infection with HHV-6 or HHV-7 by flow cytometry. Among surface molecules examined, CD3 expression appeared to decline markedly after infection with HHV-6 variant A (strain U1102) but the decreased level of CD3 expression after infection with HHV-6 variant B (strain Z29) was slight. Impairment of surface CD3 expression on HHV-6 variant A-infected cells was also demonstrated by measuring intracellular free Ca2+ concentration in response to anti-CD3 mAb. In contrast, HHV-7 infection induced a marked loss of surface CD4 expression, but the decline of CD3 expression was slight. Cytotoxic activity of virus-specific CD4+ CTL clones decreased after infection with both HHV-6 variant A or HHV-7 but the degree of reduction of cytotoxicity by HHV-6 variant B was not significant. Addition of lectin restored the cytotoxicity of HHV-7-infected CTL but not that of HHV-6 variant A-infected CTL. Northern blot analysis and immunoprecipitation showed that infection with HHV-6 and HHV-7 did not affect the transcription and protein synthesis of CD3 and CD4. These findings suggest that both HHV-6 and HHV-7 may directly cause T cell immunodeficiency but that the mechanisms of CD4+ T cell dysfunction mediated by HHV-6 and HHV-7 are different.

Specificity analysis of human CD4+ T-cell clones directed against human herpesvirus 6 (HHV-6), HHV-7, and human cytomegalovirus.

In order to clarify antigenic variations among various isolates of human herpesvirus 6 (HHV-6) and cross-reactivity among HHV-6, HHV-7, and human cytomegalovirus (HCMV) in the T-cell immune response, the antigenic specificity of the proliferative response mediated by 232 CD4+ human T-cell clones directed against HHV-6, HHV-7, or HCMV was examined. The results obtained were as follows. (i) Although the majority of T-cell clones directed against HHV-6 proliferated in response to stimulation with all strains of HHV-6 used (U1102, Z29, SF, and HST), 7% (8 of 122) of the T-cell clones showed distinct patterns of proliferative response against strain U1102 (group A) and other strains of HHV-6 (group B). (ii) Of 99 T-cell clones, 71 showed a distinct proliferative response to HHV-6 and HHV-7, whereas 28 proliferated in response to stimulation with both HHV-6 and HHV-7. (iii) A small number of T-cell clones (9 of 232) showed cross-reactivity against HHV-6 and HCMV, and 2 of the 232 clones were reactive with HCMV as well as with HHV-6 and HHV-7. (iv) The specificity of gamma interferon production by T-cell clones following the stimulation with virus antigen was identical to that of their proliferative response. These data thus indicate the presence of antigenic variations among isolates of HHV-6 and also epitopes common to HHV-6 and HHV-7 and to HHV-6, HHV-7, and HCMV which are recognized by CD4+ T cells.