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Notch signaling is involved in tumorigenesis, but its role in B-chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor-kappa B (NF-kappaB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the gamma-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-kappaB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.

Background Aims: The impact of chronic lymphatic leukemia (CLL) tumor burden on the autologous immune system has already been demonstrated. This study attempted to elucidate the molecular mechanisms underlying T-cell immunologic deficiencies in CLL., Methods: Freshly isolated CD3(+) T cells from patients with a diagnosis of CLL and healthy donors were analyzed by gene expression profiling. Activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity against mutated and unmutated autologous B cells and DAUDI, K562 and P815 cell lines. To investigate T-cell mediated cytotoxicity in vivo, we co-transplanted OKT3-activated T lymphocytes and autologous B-cell CLL (B-CLL) cells into NOD/SCID mice., Results: Gene expression profiles of peripheral blood T cells from B-CLL patients showed 25 down-regulated, and 31 up-regulated, genes that were mainly involved in cell differentiation, proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T-cell activation. After culture, the T-cell count remained unchanged, CD8 cells expanded more than CD4 and a cytotoxicity index >30% was present in 5/20 patients. Cytotoxicity against B autologous leukemic cells did not correlate with B-cell mutational status. Only activated T cells exerting cytotoxicity against autologous leukemic B cells prevented CLL in a human-mouse chimera., Conclusions: This study indicates that patients with CLL are affected by a partial immunologic defect that might be somewhat susceptible to repair. This study identifies the molecular pathways underlying T-cell deficiencies in CLL and shows that cytotoxic T-cell functions against autologous B-CLL can be rebuilt at least in part in vitro and in vivo.

The proteasome inhibitor bortezomib may increase osteoblast-related markers in multiple myeloma (MM) patients; however, its potential osteoblastic stimulatory effect is not known. In this study, we show that bortezomib significantly induced a stimulatory effect on osteoblast markers in human mesenchymal cells without affecting the number of osteoblast progenitors in bone marrow cultures or the viability of mature osteoblasts. Consistently we found that bortezomib significantly increased the transcription factor Runx2/Cbfa1 activity in human osteoblast progenitors and osteoblasts without affecting nuclear and cytoplasmatic active beta-catenin levels. Consequently a stimulatory effect of bortezomib on bone nodule formation was also demonstrated in osteoblast progenitors. These in vitro observations were confirmed in vivo by the finding of a significant increase in the number of osteoblastic cells x mm(2) of bone tissue and in the number of Runx2/Cbfa1-positive osteoblastic cells that was observed in MM patients who responded to bortezomib. Our in vitro and in vivo observations support the hypothesis that a direct stimulatory effect on bone formation process could occur during bortezomib treatment.

Consolidated evidence highlights the importance of redox signalling in poising the balance between self-renewal and differentiation in adult stem cells. The present study shows that human hematopoietic stem/progenitor cells (HSCs) constitutively generate low levels of hydrogen peroxide whose production is inhibited by DPI, apocynin, catalase, and LY294002 and scarcely stimulated by PMA. Moreover, it is shown that HSCs express at the mRNA and protein levels the catalytic subunits of NOX1, NOX2, and NOX4 isoforms of the NADPH oxidase family along with the complete battery of the regulatory subunits p22, p40, p47, p67, rac1, rac2, NOXO1, and NOXA1 as well as the splicing variant NOX2s and that the three NOX isoforms are largely co-expressed in the same HSC. These findings are interpreted in terms of a positive feed-back mechanism of NOXs activation enabling a fine tuning of the ROS level to be possibly used in redox-mediated signalling for growth and differentiation of HSCs.

We found that MEK1 inhibitor PD184352 strikingly increased apoptosis induced by arsenic trioxide (ATO) in 21 of 25 patients with primary acute myelogenous leukemia (AML). Isobologram analysis confirmed the synergistic (13 of 25 patients) or additive (8 of 25 patients) nature of this interaction. Moreover, we demonstrated that the p53-related gene p73 is a molecular target of the combined treatment in AML blasts. Indeed, ATO modulates the expression of the p73 gene by inducing the proapoptotic and antiproliferative TAp73 and the antiapoptotic and proproliferative DeltaNp73 isoforms, thereby failing to elevate the TA/DeltaNp73 ratio. Conversely, treatment with PD184352 reduces the level of DeltaNp73 and blunts the arsenic-mediated up-regulation of DeltaNp73, thus causing an increase in the TA/DeltaNp73 ratio of dual-treated cells. High doses of ATO induced p53 accumulation in 11 of 21 patients. Combined treatment resulted in the induction of the proapoptotic p53/p73 target gene p53AIP1 (p53-regulated apoptosis-inducing protein 1) and greatly enhanced the apoptosis of treated cells.

The Gruppo Italiano Malattie Ematologiche dell'Adulto (GIMEMA) 0496 protocol, through the central handling of bone marrow samples at presentation, allowed us to combine cytogenetic and molecular information on a large series of adults with acute lymphoblastic leukemia (ALL) treated homogeneously, enabling us to define as broadly as possible their genetic profile and to determine the impact on outcome of the cytogenetic-molecular signature. Of 414 patients centrally processed, 325 were considered for the categorization into the following cytogenetic-molecular subgroups: normal, t(9;22)/BCR-ABL, t(4;11)/MLL-AF4, t(1;19)/E2A-PBX1, 9p/p15-p16 deletions, 6q deletions, miscellaneous structural abnormalities, and hyperdiploid. The inclusion into each subgroup was based on a hierarchical approach: molecular abnormalities with adverse prognosis had precedence over karyotypic changes with less-defined prognosis and the latter over ploidy. Patients without abnormalities and those with isolated 9p/p15-p16 deletions showed a relatively favorable outcome (median disease-free survival [DFS], > 3 years). The t(9;22)/BCR-ABL, t(4;11)/MLL-AF4, t(1; 19)/E2A-PBX1 defined a group with dismal prognosis (median DFS, 7 months), whereas 6q deletions, miscellaneous aberrations, and hyperdiploidy predicted an intermediate prognosis (median DFS, 19 months). This study highlights the importance of a combined cytogenetic-molecular profiling of adult ALL at presentation as a critical independent determinant of their outcome, providing further evidence of the necessity of a risk-adapted therapeutic algorithm for an optimal management of these patients.

Establishment of cell lines capable of killing leukemia cells, in the absence of alloreactivity against normal host cells, represents a most desirable goal in bone marrow transplantation (BMT) and cancer immunotherapy. By using a human --> mouse chimeric model, we demonstrate that allogeneic anti-third-party cytotoxic T lymphocytes (CTLs) depleted of alloreactivity are endowed with a potent anti-B-cell chronic lymphocytic leukemia (B-CLL) reactivity. Likewise, CTL preparations generated from autologous T cells of the same patients with B-CLL exhibited comparable leukemia eradication, suggesting that the reactivity of allogeneic anti-third-party CTLs is not mediated by residual antihost clones. This specificity was also exhibited in vitro, and annexin staining revealed that B-CLL killing is mediated by apoptosis. While the CTLs killing of third-party cells could be blocked by anti-CD3 antibody, the lysis of the B-CLL cells was not inhibited by this antibody, suggesting a T-cell receptor (TCR)-independent cytotoxicity. The role of cell contact leading to apoptosis of B-CLL cells is shown in transwell plates and by anti-lymphocyte function-associated antigen-1 (LFA-1)-blocking antibody. Up-regulation of CD54 and the subsequent apoptosis of B-CLL cells depend on the initial LFA-1/ICAM-1 (intercellular adhesion molecule 1) interaction. Taken together, these results suggest that allogeneic or autologous host nonreactive anti-third-party CTLs may represent a new therapeutic approach for patients with B-CLL.

Previous studies suggest that cells within the CD34(+) hematopoietic stem cell compartment are endowed with immune regulatory activity. Furthermore, it is possible to expand the human regulatory cells upon short-term culture of purified CD34+ cells with an early-acting cytokine cocktail. We now show that addition of anti-CD28, anti-CD2, interleukin-2 (IL-2), anti-IL-10, or IL-12 to the bulk mixed lymphocyte reaction (MLR) cannot reverse the inhibitory activity of the CD34+ cells, ruling out anergy-based mechanisms or mechanisms involving Th1-Th2 skewing. Furthermore, phenotyping of cells present after addition of CD34+ cells to the bulk MLR ruled out potential induction of plasmacytoid dendritic precursors, known to be endowed with regulatory activity. In contrast, the inhibitory activity of CD34+ cells could be reversed by adding the caspase inhibitor BD-FMK to the bulk MLR, indicating a deletion-based mechanism. The deletion can be inhibited by anti-tumor necrosis factor-alpha (anti-TNF-alpha) and not by anti-transforming growth factor-beta (anti-TGF-beta), suggesting a potential role for TNF-alpha in the regulatory activity of CD34+ cells.

Background: Although imatinib is the standard treatment for chronic myeloid leukaemia, not all patients reach complete cytogenetic remission (CCR) and most maintain detectable disease at the molecular level. We investigated whether a vaccine targeting the BCR-ABL-derived p210 fusion protein was an active and specific immunotherapy., Methods: We recruited 16 patients who had chronic myeloid leukaemia (with the b3a2 fusion point of p210), stable residual disease, a minimum treatment of 12 months of imatinib or 24 months of interferon alfa, and no further reduction of residual disease for at least 6 months preceding enrollment. They were given six vaccinations with a peptide vaccine derived from the sequence p210-b3a2 plus molgramostim and QS-21 as adjuvants (CMLVAX100) before assessment of immunological and disease response, which included detecting amounts of b3a2 transcripts by standardised quantitative real-time reverse-transcriptase PCR., Results: Of ten patients on imatinib, nine started CMLVAX100 having had a median of 10 months' stable cytogenetic disease (median 10% Philadelphia-chromosome-positive metaphases), whereas one started in stable CCR. All patients' cytogenetic responses improved after six vaccinations, with five reaching CCR. Notably, three of these five patients also had undetectable amounts of b3a2 transcript (BCR-ABL:beta2 microglobulin ratio <0.00001). Six patients on interferon alfa treatment with a median of 17 months' stable residual disease (median 13% Philadelphia-chromosome-positive cells) were also vaccinated. All but one had improved cytogenetic responses, and two reached CCR. Overall, we recorded peptide-specific delayed-type hypersensitivity (in 11 of 16 patients), CD4 cell proliferation (13 of 14 assessed), and interferon gamma production (five of five assessed)., Interpretation: Addition of CMLVAX100 to conventional treatment in patients with chronic myeloid leukaemia might favour further reduction of residual disease and increase the number of patients reaching a molecular response.

Endothelial cells (EC) were extracted through a lectin-based method from bone marrow of 57 patients with active multiple myeloma (MM) and compared with their healthy quiescent counterpart, human umbilical vein EC (HUVEC). MMECs exhibit specific antigens that indicate ongoing angiogenesis and embryo vasculogenesis; solid intercellular connections, hence stability of MM neovessels; and frequent interactions with plasma cells, hence tumor dissemination. They show heterogeneous antigen expression, hence existence of subsets. Their main genetic markers are indicative of a vascular phase. They show intrinsic angiogenic ability, because they rapidly form a capillary network in vitro, and extrinsic ability, because they generate numerous new vessels in vivo. They vividly secrete growth and invasive factors for plasma cells. They signal through kinases mandatory for development of neovascularization. Ultrastructurally, they are abnormal and show metabolic activation, like tumor ECs. Thalidomide heavily interferes with their functions. Vasculogenesis and angiogenesis might contribute to the MM vascular tree and progression, in the form of growth, invasion, and dissemination. In view of the heterogeneity of the antigenic phenotype of MMECs, a mixture (or a sequence) of antiangiogenic agents coupled with thalidomide would seem plausible for the biologic management of MM.

In mammals, methylation of DNA within regulatory sites and histone deacetylase recruitment in transcriptional repressing domains are involved in the loss of the expression of retroviral DNA or repeat arrays transferred in cells for therapeutic purposes. Various investigation results suggest that methylation/deacetylation events are modulated by extracellular and cytoplasmic signal transduction pathways closely involved in regulating cell differentiation. To analyse gene silencing mechanisms and assess if potential pharmacological treatment affects gene silencing kinetics we transduced U937 myelomonocytic cells with a bicistronic retroviral construct carrying the herpes simplex virus thymidine kinase (HSV-TK) and beta-galactosidase (Lac-Z) genes. This vector can be employed in vivo and in vitro to render transduced cell populations susceptible to ganciclovir (GCV). We verified the effect of the histone deacetylase inhibitor Trichostatin A (TSA) alone or combined with 5'-azacytidine (5'aza-C) on transcription downmodulation. Our results indicate that in our in vitro model TSA is able to reactivate transgene expression, more efficiently and with quicker kinetics (12-24h) than 5'aza-C (36-48 h). The effect is dose dependent (between 1 and 50 nM), with no relevant toxicity. Treatment with both drugs is synergistic in gene reactivation in terms of extension and persistence, with low toxicity and no relevant differentiating effects. The cells in which transgene expression has been reactivated undergo progressive silencing, but once weekly drug treatment can maintain high transgene expression levels for more than 90 days with no evidence of selection. The results obtained by treating U937 transduced clones with TSA and/or 5'aza-C together with IL-3, G-CSF or GM-CSF cytokines suggest that transduced U937 differentiation levels do not affect basal expression, but render these cells more responsive to reactivation by TSA or TSA plus 5'aza-C, but not to 5'aza-C alone. In conclusion, the results suggest that in vitro inhibition of histone deacetylase by TSA can interfere with gene silencing mechanisms affecting 5' Moloney murine leukaemia virus long terminal repeat (MoMuLV-LTR) driven transgene expression thus providing the rationale for TSA and/or 5'aza-C administration in animal models for the translation on gene therapy applications.

Stem cell-dose escalation is one way to overcome immune rejection of incompatible stem cells. However, the number of hematopoietic precursors required for overcoming the immune barrier in recipients pretreated with sublethal regimens cannot be attained with the state-of-the-art technology for stem cell mobilization. This issue was addressed by the observation that cells within the human CD34(+) population are endowed with veto activity. In the current study, we demonstrated that it is possible to harvest about 28- to 80-fold more veto cells on culturing of purified CD34(+) cells for 7 to 12 days with an early-acting cytokine mixture including Flt3-ligand, stem cell factor, and thrombopoietin. Analysis of the expanded cells with fluorescence-activated cell-sorter scanning revealed that the predominant phenotype of CD34(+)CD33(-) cells used at the initiation of the culture was replaced at the end of the culture by cells expressing early myeloid phenotypes such as CD34(+)CD33(+) and CD34(-)CD33(+). These maturation events were associated with a significant gain in veto activity as exemplified by the minimal ratio of veto to effector cells at which significant veto activity was detected. Thus, whereas purified unexpanded CD34(+) cells exhibited veto activity at a veto-to-effector cell ratio of 0.5, the expanded cells attained an equivalent activity at a ratio of 0.125. The availability of novel sources of veto cells such as those in this study might contribute to the realization of immunologic tolerance in "minitransplants," without any risk of graft-versus-host disease.

The GIMEMA ALL 0288 trial was designed to evaluate the impact of a 7-day prednisone (PDN) pretreatment on complete remission (CR) achievement and length, the influence of the addition of cyclophosphamide (random I) to a conventional 4-drug induction on CR rate and duration, and whether an early post-CR intensification (random II) by an 8-drug consolidation could improve CR duration. Median follow-up of this study was 7.3 years. From January 1988 to April 1994, among 794 adult (> 12 but < 60 years) patients registered, 778 were eligible. Their median age was 27.5 years; 73% had B-lineage acute lymphoblastic leukemia (ALL) and 22% had T-lineage disease; 18% showed associated myeloid markers; 47 of 216 analyzed patients (22%) had Philadelphia chromosome-positive ALL. Response to PDN pretreatment was observed in 65% of cases. CR was achieved in 627 patients (82%). Resistant patients and induction death rates were 11% and 7%, respectively. Random II was applied to 388 patients with CR; 201 had maintenance alone and 187 had consolidation followed by maintenance. The relapse rate was 60%; isolated central nervous system relapses were 8% of all CRs and 13% of all relapses. Median survival (overall survival [OS]), continuous complete remission (CCR), and disease-free survival (DFS) were 2.2, 2.4, and 2 years, respectively. PDN pretreatment response resulted the main independent factor influencing CR achievement, OS, CCR, and DFS; the addition of cyclophosphamide in induction significantly influenced CR achievement in a multivariate analysis. Neither induction intensification nor early consolidation appeared to influence CCR and DFS duration. For the first time PDN pretreatment response proved to be a powerful factor predicting disease outcome in adult ALL patients.

In acute leukemia patients, infusing a megadose of extensively T-cell-depleted hematopoietic stem cells after an immuno-myeloablative conditioning regimen ensures sustained engraftment of full-haplotype mismatched transplants without graft-vs-host disease. Besides the conditioning regimen and the megadose of stem cells donor natural killer cell alloreactivity also plays a role in facilitating engraftment and in preventing relapse. Since our first successful pilot study, our efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the post-transplant immunological recovery. The results we have so far achieved in 112 very high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype mismatched donor, who is immediately available, a T-cell depleted mismatched transplant should be offered, not as a last resort, but as a viable option to high risk acute leukemia patients who do not have, or cannot find, a matched donor., (Copyright 2001 Harcourt Publishers Ltd.)

In human leukocyte antigen haplotype-mismatched transplantation, extensive T-cell depletion prevents graft-versus-host disease (GVHD) but delays immune recovery. Granulocyte colony-stimulating factor (G-CSF) is given to donors to mobilize stem cells and to recipients to ensure engraftment. Studies have shown that G-CSF promotes T-helper (Th)-2 immune deviation which, unlike Th1 responses, does not protect against intracellular pathogens and fungi. The effect of administration of G-CSF to recipients of mismatched hematopoietic transplants with respect to transplantation outcome and functional immune recovery was investigated. In 43 patients with acute leukemia who received G-CSF after transplantation, the engraftment rate was 95%. However, the patients had a long-lasting type 2 immune reactivity, ie, Th2-inducing dendritic cells not producing interleukin 12 (IL-12) and high frequencies of IL-4- and IL-10-producing CD4(+) cells not expressing the IL-12 receptor beta(2) chain. Similar immune reactivity patterns were observed on exposure of donor cells to G-CSF. Elimination of postgrafting administration of G-CSF in a subsequent series of 36 patients with acute leukemia, while not adversely affecting engraftment rate (93%), resulted in the anticipated appearance of IL-12-producing dendritic cells (1-3 months after transplantation versus > 12 months in transplant recipients given G-CSF), of CD4(+) cells of a mixed Th0/Th1 phenotype, and of antifungal T-cell reactivity in vitro. Moreover, CD4(+) cell counts increased in significantly less time. Finally, elimination of G-CSF-mediated immune suppression did not significantly increase the incidence of GVHD (< 15%). Thus, this study found that administration of G-CSF to recipients of T-cell-depleted hematopoietic transplants was associated with abnormal antigen-presenting cell functions and T-cell reactivity. Elimination of postgrafting administration of G-CSF prevented immune dysregulation and accelerated functional immune recovery.

The engraftment capacity of bone marrow-derived mesenchymal cells was investigated in 41 patients who had received a sex-mismatched, T-cell-depleted allograft from human leukocyte antigen (HLA)-matched or -mismatched family donors. Polymerase chain reaction (PCR) analysis of the human androgen receptor (HUMARA) or the amelogenin genes was used to detect donor-derived mesenchymal cells. Only 14 marrow samples (34%) from 41 consenting patients generated a marrow stromal layer adequate for PCR analysis. Monocyte-macrophage contamination of marrow stromal layers was reduced below the levels of sensitivity of HUMARA and amelogenin assays (5% and 3%, respectively) by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. Patients who received allografts from 12 female donors were analyzed by means of the HUMARA assay, and in 5 of 12 cases a partial female origin of stromal cells was demonstrated. Two patients who received allografts from male donors were analyzed by amplifying the amelogenin gene, and in both cases a partial male origin of stromal cells was shown. Fluorescent in situ hybridization analysis using a Y probe confirmed the results of PCR analysis and demonstrated in 2 cases the existence of a mixed chimerism at the stromal cell level. There was no statistical difference detected between the dose of fibroblast progenitors (colony-forming unit-F [CFU-F]) infused to patients with donor- or host-derived stromal cells (1.18 +/- 0.13 x 10(4)/kg vs 1. 19 +/- 0.19 x 10(4)/kg; P >/=.97). In conclusion, marrow stromal progenitors reinfused in patients receiving a T-cell-depleted allograft have a limited capacity of reconstituting marrow mesenchymal cells.

The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia (Ph) chromosome that fuses genetic sequences of the BCR gene on chromosome 22 with c-ABL sequences translocated from chromosome 9. BCR/ABL fusion proteins have a dysregulated protein tyrosine kinase (PTK) activity exerting a key role in malignant transformation. Targeting the tyrosine kinase activity of BCR/ABL or using agents capable of triggering apoptosis might represent attractive therapeutic approaches for ex vivo purging. AG957, a member of the tyrphostin compounds, exerts a selective inhibition of p210(BCR/ABL) tyrosine phosphorylation. We report here that preincubation of CML or normal CD34(+) cells with graded concentration of AG957 (1 to 100 micromol/L) resulted in a statistically significant, dose-dependent suppression of colony growth from multipotent, erythroid, and granulocyte-macrophage progenitors as well as the more primitive long-term culture-initiating cells (LTC-IC). However, AG957 doses causing 50% inhibition (ID50) of CML and normal progenitors were significantly different for multilineage colony-forming units (CFU-Mix; 12 v 64 micromol/L; P =.008), burst-forming unit-erythroid (BFU-E; 29 v 89 micromol/L; P =.004), colony-forming unit-granulocyte-macrophage (CFU-GM; 34 v 85 micromol/L; P =.004), and LTC-IC (43 v 181 micromol/L; P =.004). In 5 of 10 patients, analysis of BCR/ABL mRNA on single progenitors by reverse transcription-polymerase chain reaction showed that AG957 at 50 micromol/L significantly reduced the mean (+/-SD) percentage of BCR/ABL-positive progenitors (92% +/- 10% v 33 +/- 5%; P =.001). Because AG957 treatment resulted in significantly higher percentages of apoptotic cells (30% v 9%) in the BCR/ABL-transfected 32DLG7 cells as compared with 32D-T2/93 cells (BCR/ABL-negative), we investigated the combined effects of AG957 with the anti-Fas receptor (Fas-R) monoclonal antibody CH11 that triggers apoptosis. As compared with AG957 alone, the sequential treatment of CML CD34(+) cells with AG957 (1 micromol/L) and CH11 (1 microgram/mL) increased CFU-Mix, BFU-E, and CFU-GM growth inhibition by 1.6-fold, 3-fold, and 4-fold, respectively. In contrast, the treatment of normal CD34(+) cells with AG957 and CH11 failed to enhance AG957-induced colony growth inhibition. We conclude that (1) AG957 inhibits in a dose-dependent manner CML CD34-derived colony formation by both primitive LTC-IC as well as committed CFU-Mix, BFU-E, and CFU-GM; (2) this growth inhibition is associated with the selection of a substantial amount of BCR/ABL-negative progenitors; and (3) the antiproliferative effect of AG957 is dramatically increased by combining this compound with the anti-Fas-R antibody CH11. These data may have significant therapeutic applications.

Patients who undergo transplantation with haploidentical "three-loci" mismatched T-cell-depleted bone marrow (BM) are at high risk for graft failure. To overcome the host-versus-graft barrier, we increased the size of the graft inoculum, which has been shown to be a major factor in controlling both immune rejection and stem cell competition in murine models. Seventeen patients (mean age, 23.2 years; range, 6 to 51 years) with end-stage chemoresistant leukemia were received transplants of a combination of BM with recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells from HLA-haploidentical "three-loci" incompatible family members. The average concentration of colony-forming unit-granulocyte-macrophage in the final inoculum was sevenfold to 10-fold greater than that found in BM alone. The sole graft-versus-host disease (GVHD) prophylaxis consisted of T-cell depletion of the graft by the soybean agglutination and E-rosetting technique. The conditioning regimen included total body irradiation in a single fraction at a fast dose rate, antithymocyte globulin, cyclophosphamide and thiotepa to provide both immunosuppression and myeloablation. One patient rejected the graft and the other 16 had early and sustained full donor-type engraftment. One patient who received a much greater quantity of T lymphocytes than any other patient died from grade IV acute GVHD. There were no other cases of GVHD > or = grade II. Nine patients died from transplant-related toxicity, 2 relapsed, and 6 patients are alive and event-free at a median follow-up of 230 days (range, 100 to 485 days). Our results show that a highly immunosuppressive and myeloablative conditioning followed by transplantation of a large number of stem cells depleted of T lymphocytes by soybean agglutination and E-rosetting technique has made transplantation of three HLA-antigen disparate grafts possible, with only rare cases of GVHD.

FLT3, a receptor belonging to the FMS/KIT family and localized to 13q12, could play a role in the biology of early hematopoietic progenitor cells. Because FMS and KIT are expressed in both normal progenitors and myeloid leukemias, we looked for FLT3 expression in fresh human leukemic cells using Northern blot analysis. High levels of FLT3 expression were detected in 92% of the cases of acute myeloid leukemia (AML) tested, ranging from the M1 to the M5 stages of differentiation assessed in the French-American-British classification. Immature (MO) AML cells, biphenotypic leukemias, and AML with megakaryocytic differentiation (M7 subtype) also expressed the FLT3 transcript. FLT3 was also expressed at high levels in acute lymphoid leukemias of T and B origins. Finally, it was not expressed in chronic myeloid leukemias in chronic phase, whereas it was expressed in most blast crisis samples. This pattern of expression of FLT3 contrasts with the expression of FMS and KIT restricted to myeloid leukemias, and suggests that the FLT3 product could play a role in the expansion of the leukemic blasts of both the myeloid and lymphoid lineages.

V pre-B and lambda-like genes are selectively expressed in human pre-B cells and encode polypeptide chains that associate in a mu-pseudolight chain complex that may regulate some crucial steps of early B-cell differentiation. We have followed by polymerase chain reaction and Northern blot analysis the expression of these "pre-B-specific" genes in correlation with the status (rearranged v germline) of Ig gene loci (H, kappa, lambda) in a panel of 32 leukemias pertaining mostly to the B lineage and including a number of ambiguously characterized samples. All cells that had rearranged the H locus only expressed V pre-B and lambda-like transcripts, in agreement with a pre-B status. In this group, some biphenotypic leukemias (mostly My/B) might, in fact, be already engaged in the B lineage. Rearrangement of V kappa or V lambda loci correlated with the disappearance of the pre-B gene products. In a pre-B acute lymphoblastic leukemia cell line that was induced to mature to the B-cell stage in culture upon kappa gene rearrangement, the mu-pseudolight chain complex was actually replaced by the classical mu-kappa molecule. Finally, V pre-B and lambda-like genes were found expressed in two leukemic cells that had retained all Ig loci in germline configuration. This finding raises the possibility of having an early pro-B progenitor in which V pre-B and lambda-like products associate with a H chain surrogate in a complex that would trigger an early event of B-cell differentiation such as the H locus rearrangements.

The effect of various phorbols and phorbol diesters on the NK sensitivity of the human leukemic K562 cells was studied. A marked decrease in K562 cell susceptibility was achieved by culture in the presence of either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or beta-phorbol-dibutyrate. The maximum protection against NK lysis was achieved when K562 cells were cultured in the presence of 160 nM TPA for 48 hr (mean percentage inhibition: 61% of specific lysis). As for untreated targets, the residual killing of K562 cells after TPA treatment was mediated through large granular lymphocytes (LGL). The experimental procedures required to achieve maximal NK protection with TPA resulted simultaneously in marked phenotypical changes in K562 cells: erythroid and early myeloid markers decreased, whereas the expression of megakaryocytic markers was increased as shown by staining with antiplatelet monoclonal antibodies and assessment of platelet peroxidase activity. Chemical phorbol analogs which were unable to induce K562 cell differentiation did not affect K562 cell sensitivity to NK lysis. De novo protein synthesis is involved in the TPA-induced NK resistance, since this effect was abolished by pretreatment of K562 cells with actinomycin D or cycloheximide. TPA has been previously demonstrated to reduce NK effector activity. In our data however, the observed TPA effects were not due to release of TPA acting on effector cells during the NK assay since TPA-treated K562 cell supernatants were unable to inhibit NK activity in control assays. Thus, TPA appears to decrease NK killing of malignant cells, both by depressing NK effector cells functions and by reducing the susceptibility to NK lysis of the target cells. In single-cell agarose assays, TPA-treated K562 cells demonstrated reduced NK-binding capacity and reduced sensitivity to lysis after binding. These defects could not be reversed by activation of the NK effector cells with interferon. The results here reported extend the previously suggested relations between the expression of NK-target structures and the differentiation stage of malignant cells.

A 14q+ marker with extra material derived from chromosome 11 long arm, i.e. segment q13----qter, has been found in cells from a pleural effusion in a patient with highly malignant multiple myeloma. The segment 11q13----qter was trisomic because of the presence of both apparently normal homologous chromosomes 11.

Staphylococcal protein A conjugated to horseradish peroxidase was employed in an indirect immuno-staining technique to identify intracellular antigens in paraffin-embedded tissues. The sections were incubated with specific antisera and the antigen-IgG complexes demonstrated with protein A-peroxidase conjugate. Immunoglobulins, lysozyme and insulin were satisfactorily detected by this technique. A comparison of this method with the PAP, "labelled antigen" and peroxidase-labelled antibody sandwich techniques was made.

The phenotype of fresh and cultured leukemic cells from patients with hairy cell leukemia was studied using a panel of monoclonal antibodies in addition to the detection of peroxidase activity under electron microscopy. In fresh samples, the leukemic cells from 11 patients displayed predominantly a B phenotype, as judged by their reactivity with the B1 monoclonal antibody and surface immunoglobulin expression. Ultrastructural peroxidase activity, characteristic of hairy cells, was observed in all cases studied. When hairy cells were cultured in the presence of phytohemagglutinin and irradiated T cells, their phenotype converted from surface Ig+, B1+, OKT3-, OKT11- to surface Ig-, B1+, OKT3-, OKT11+. In contrast, the peroxidase activity remained unchanged. Some hairy cells were also OKM1+, but no conclusion could be made about the MO2 antigen, a more specific marker of monocytes. The variability of the phenotype in vivo and in vitro indicates that reliable markers are required for identifying hairy cells. When studied together, the staining by B1 monoclonal antibody and the ultrastructural detection of peroxidase, enable the identification of hairy cells with certainty.

Since vimentin intermediate filament (IF) expression in hemopoietic cells varies with the cell lineage as well as the state of differentiation of the cells, we studied the vimentin cytoskeleton by direct immunofluorescence and electron microscopy in 50 cases of acute nonlymphocytic leukemias. We found that malignant cells tend to reproduce the vimentin organization characteristic of their normal cellular counterpart. Thus, in M2 and M3 leukemias (French-American-British classification), vimentin was often reduced to a juxtanuclear bundle of filaments contrasting with the rich filamentous network expressed by M4 or M5 leukemias. In erythroblastic leukemias (M6) and megakaryoblastic leukemias, both identified by the expression of lineage-specific antigens, the absence of vimentin IFs could be correlated with the level of differentiation reached by the blasts. M1 leukemias displayed an abnormal pattern of vimentin organization with aggregated filaments giving a ring-like structure. However, no abnormality of the vimentin polypeptide could be detected by two-dimensional electrophoresis. These results show that the expression of the vimentin IF cytoskeleton may be a useful marker of differentiation in the study of leukemic cells.