ALBERT

Description: Umbilical cord is an extra-embryonic-annex rich of both hematopoietic stem and progenitor cells (HSPC) and mesenchymal stem cells (MSC) and it is easily accessible. The HSPC derived from umbilical cord blood (UCB) are promising as graft for allogeneic bone marrow (BM) transplantation and as source of target cells for autologous HSPC gene correction. UCB-HSPC have several advantages compared to adult ones: a less risk of graft-versus-host disease, a higher frequency of progenitors with a greater clonogenic potential and more susceptibility to be transduced by lentiviral vectors. Nonetheless, the HSPC yield from single cord blood unit is not sufficient for these clinical approaches in adults. Therefore, ex-vivo expansion of HSPC in media supplemented by cytokines and/or in vitro culture systems with feeder layers, is a valid approach to exceed this limit. MSC are a component of BM-microenvironment that play a key role in supporting of hematopoiesis by ability to secrete soluble factors and probably by the direct cell-cell interaction too. In this work, we investigated the ability of umbilical cord extracellular matrix-MSC (Wharton's Jelly-MSC) to support the ex-vivo expansion of UBC- purified CD34+ cells. In particular, we evaluated the fold increase, and the frequency of CD34+ cell and CD34+subtypes during expansion at the following culture conditions: by direct contact with WJ-MSC layer, by exposure to the soluble factors secreted by WJ-MSC layer in transwell system. The fold expansion was compared with the CD34+ cells expanded in a customized serum-free medium. CD34+ cells were isolated by immuneselection from 8 fresh UCB. The WJ-MSC were isolated from UC cut-pieces by non-enzymatic procedure but thanks to their capacity to migrate to plastic substrate. At the confluence of 60-70% the WJ-MSC were treated with mytomicin-C to arrest the cell cycle. After 48h, the immune-selected CD34+ cells were seeded in WJ-MSC at the density of 5-10 x104 in 12 well plates by direct or indirect contact (by transwell system). CD34+cells were grown in absence of feeder layers at the same conditions. Early hematopoietic cytokines (Flt-3, TPO, SCF) were supplemented in all three conditions and freshly replaced every two days of culture. Numbers and frequency of CD34+cells were evaluated according to ISHAGE method and CD34+ subtyping was performed by four color method to investigated the co-expression of the primitive surface antigens (CD38, CD133, CD90). The frequency of CD34+ cells at day 5 of culture decreased only 10% and was about 50% after 8 days of culture in conditions. The expansion of CD34 + cells at direct contact with WJ-MSC was superior (5.5 fold increase) compared to that of the other two conditions (3 fold on average). At day 8of culture, the CD34+ cells expanded 12 fold at direct contact with feeder layer, about 7 fold in a transwell system and 6 fold in basic medium. No substantial differences in the grade of expansion was revealed in heterologous vs homologous co-cultures of HSPC/WJ-MSC. Noteworthy is that in the contact system in addition to the fluctuating CD34+ cells harvested from the medium (floating CD34+ cells), we found approximately 50% of the total CD34+ cells be adherent to WJ-MSC layer, these cells were released only after enzymatic proteolytic treatment. Subtyping the CD34+cell population growing in contact to the WJ-MSC or in the conditioned medium we found that the CD34+/CD133+cell population was maintained high (72% ±12 over the total CD34+ cells) as in unmanipulated CB-HSC. The CD34+CD38- cells decreased by 2,5 fold in both systems, as early as day 5 of culture. However, in the contact system this population was 3 times more represented in the attached CD34+ cell fraction. The CD34+/CD90+ subtype was also expanded (more than 8 fold) particularly in the attached fraction, as early as 5 days of culture and was maintained to the end WJ-MSC supported ex-vivo HSPC expansion with superior effect in a cell contact system. Two phenotypically different populations of HSPC developed in this system with an increased frequency of CD34+ cells that co-expressed markers typical of more early progenitors in the attached CD34+ cell fraction. We are assessing the significance of these differences by performing molecular and functional studies of WJ-MSC-supported HSPC. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F12000150004 Disclosures No relevant conflicts of interest to declare.

Description: Ex vivo expansion of hematopoietic stem/progenitor cells may result in defective engraftment. Human cord blood CD34+ progenitor cells were synchronized and assayed for adhesion and migration onto fibronectin (Fn) and vascular cell adhesion molecule-1 (VCAM-1) at different stages of a first cell cycle executed ex vivo. During S phase transit, adhesion to Fn was transiently increased while binding to VCAM-1 was reversibly decreased, after which adhesion to both ligands returned to baseline levels with cell cycle completion. Transmigration across Fn and VCAM-1 decreased irreversibly during S phase progression. The function of α4 and α5 integrins was assessed with specific neutralizing antibodies. In uncultured CD34+ cells and long-term culture-initiating cells (LTC-ICs), both adhesion and migration on Fn were inhibited by anti-α4 but not by anti-α5 antibodies. In mitotically activated CD34+ cells and LTC-ICs, adhesion and migration on Fn were mainly dependent on α5 integrin and to a lesser extent on α4 integrin. Changes in integrin function were not dependent on parallel modulation of integrin expression. In conclusion, Fn and VCAM-1 binding of progenitor cells fluctuates reversibly during cell cycle transit ex vivo. In addition, our data show that mitogenic activation induces a shift from a dominant α4 to a preferential α5 integrin–dependent interaction with Fn.

Description: Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disease driven by the Bcr-Abl hybrid protein expressed in the hemopoietic stem cell compartment. Bcr-Abl deregulated tyrosine kinase (TK) activity, impairs blood cells homeostasis leading to altered growth, homing and apoptosis. Imatinib mesylate (Glivec), an ATP competitor molecule active on Abl and Bcr-Abl TKs, is the gold standard therapy for Bcr-Abl positive CML, however long-term efficacy of the drug is unknown, and resistance has been described. In the present study we focused our attention on some members of the pyrrolo [1,2-b][1,2,5] benzothiadiazepines (PBTDs) family for their potential apoptotic activity. Coumpounds were tested on K562, NPE and ARO cell lines. After showing important apoptotic activity, the compounds were tested on CML cells from 10 patients at onset and 2 patients in blast crisis Imatinib-resistant. Our data evidenced strong activity in apoptosis induction in all patients, including patients that have developed blast crisis under Imatinib treatment. Three compounds (RS779, RS735 and RS678) were selected among the PBTDs. At first compounds were incubated with CML K562 cell line showing DNA fragmentation and the characteristic morphological features of apoptosis such as shrinkage, chromatin condensation, membrane blebbing, evaluated by electronic microscopy analysis. NPE and ARO (neoplastic thyroid cells) cell lines were also tested showing similar results. At this point we decided to test compounds on cells from CML patients. We harvested peripheral blood cells from 10 consecutive CML patients at onset, and also peripheral blood cells from 2 blast crisis evolved under Imatinib treatment. Peripheral blood samples were incubated with PBTDs for 8,16, and 24 hours at 10μM concentration in order to make a direct comparison of apoptotic potencies. Apoptotic DNA fragmentation was tested by agarose gels electrophoresis and was present in all patients generating a characteristic ladder pattern of discontinuous DNA fragments. The characteristic morphological features such as shrinkage, chromatin condensation and membrane blebbing were also evidenced. RS-678 and RS-779 caused strong dose- and time-dependent induction of apoptosis. The DNA fragmentation became apparent at 8 h and increased up to 16h. We also examined the expression of apoptosis-related genes in 6/10 patients at onset. In cells treated with (RS-678) and (RS-779) compounds, expression of pro-apoptotic bax and procaspase-3 genes were increased. The b-actin DNA band used as control, was distributed at similar levels in all samples, while expression of the anti apoptotic bcl-XL and bcl-2 genes were decreased. These results suggest that the apoptotic effect of these compounds is likely mediated through regulation of genes involved in apoptosis, and/or cell survival at the transcriptional level. Among the three compounds tested RS-678 and RS-779 were found to be the most active both in cell lines and in patient samples, whereas RS-735 had little or no effect. These findings suggest that PBTDs could be promising candidates as a novel therapeutic agent for Bcr-Abl-positive CML.

Description: Introduction: Molecular chaperones have many functions, such as protecting other proteins against aggregation, assisting in folding of nascent proteins/refolding of damaged proteins and targeting severely damaged proteins to degradation. As one of the molecular chaperones, Hsp90 functions to facilitate the folding of newly synthesized and denatured client proteins, including mutated p53, Bcr-Abl, p185ErbB2 and Raf-1. The Bcr-Abl fusion gene encodes for the p210Bcr-Abl tyrosine kinase (TK) implicated in the pathogenesis of chronic myelogenous leukemia (CML). Studies in cultured cells have identified many signal transduction pathways activated by Bcr-Abl, including activation of the Ras, MAPK, JNK/SAPK, phosphatidylinositol-3 kinase, nuclear factor-B and STAT pathways. Imatinib mesylate (imatinib IM) is a tyrosine kinase inhibitor that competitively inhibits ATP binding in the kinase domains of both the Bcr-Abl and c-Abl kinases. It has been suggested that resistance to imatinib stems from Bcr-Abl gene amplification, leading to overexpression of Bcr-Abl protein or point mutations in the Bcr-Abl gene However, several groups suggested that there might be other forms of Bcr-Abl-independent imatinib resistance Recently, it has been reported that changes in histone deacetylase (HDAC) expression in leukemic cells could be involved in mechanisms for abnormal cellular proliferation that operate through chromatin-independent pathways and thereby could lead to acquired drug resistance of the cells In the present study, we evaluated in primary leukemic blasts, obtained from chronic myelogenous leukemia patients at onset, patients in blast crisis and patients which were imatinib-resistant The espression the sirtuin members family and HSP70, HSP90 i-NOS and bcl-2 was evaluated by Nortern blot and Western blot analysis. Material and Methods: Primary leukaemia blasts We harvested primary blast rich mononuclear cells were obtained by gradient centrifugation on ficoll-hypaque of bone marrow and peripheral blood cells after obtaining appropriate informed consent. Northern blot Total RNAs from control or treated cells were isolated using Tri Reagent Aliquots of RNA were electrophoresed and blotted onto nylon membranes, that hybridized to 32P-labelled probe. Western Blot Cells were lysed and. then were centrifugated. Protein concentration was determined by the Bradford assay.. Equivalent amounts of protein loaded and electrophoresed and were transferred to nitrocellulose membranes, that were incubated with the different primary antibodies:, Result and Discussion:. In the present study, we evaluated a pattern of different gene expression by Northern Blot and Western Blot analysis in bone marrow and peripheral blood cells from 16 CML patients at onset, from 2 patients in blast crisis evolved under IM treatment, and 14 imatinib-resistant patients. Some RNAs were underexpressed in most or all samples tested and never overexpressed (eg SIRT2, SIRT3, SIRT4 and SIRT5), while others were overexpressed in the great majority of samples and rarely, if ever, underexpressed (eg SIRT1, SIRT7, HSP90, iNOS)Furthermore, we examined the level of heat-shock related proteins HSP90 and bcl-2 in 2 patients during treatment with IM. and one patient IM-resistant by western Blot analysis: HSP90 and BCl-2 increased one patient during treatment with IM, while both protein levels was very high in one one patient IM-resistant These results suggest that the difference of genes expression might contribute to patterns of clinical response.

Description: The early emergence of macrophages and their large pattern of tissue distribution during development suggest that they may play a critical role in the initial steps of embryogenesis. In the present study, we show that monocytic cells derived from human embryonic stem cells (hESCs) and from fetal liver follow a differentiation pathway different to that of adult cells, leading to specific functions. Embryonic and fetal monocytic cells differentiated from a CD14lowCD16− precursor to form CD14highCD16+ cells without producing the CD14highCD16− cell population that predominates in adult peripheral blood. Both demonstrated an enhanced expression of genes encoding tissue-degrading enzymes, chemokines, and scavenger receptors, as was previously reported for M2 macrophages. Compared with adult blood monocytes, embryonic and fetal monocytic cells secreted high amounts of proteins acting on tissue remodeling and angiogenesis, and most of them expressed the Tie2 receptor. Furthermore, they promoted vascular remodeling in xenotransplanted human tumors. These findings suggest that the regulation of human fetal and embryonic monocytic cell differentiation leads to the generation of cells endowed mainly with anti-inflammatory and remodeling functions. Trophic and immunosuppressive functions of M2-polarized macrophages link fetus and tumor development, and hESCs offer a valuable experimental model for in vitro studies of mechanisms sustaining these processes.

Description: Reactivation of hepatitis B virus infection in subjects receiving cytotoxic treatment for haematological malignancies occurs in 21–53% of chronic HBsAg carriers and in an unknown number of HBsAg negative subjects harbouring occult HBV infection. Immunotherapy with alemtuzumab, a humanized monoclonal antibody against CD52 on lymphoid cells, produces deep immunosuppression. We describe two subjects with chronic lymphocytic leukaemia and occult HBV infection who developed a virological and biochemical flare of hepatitis B following immunotherapy with alemtuzumab. One of them developed a full blown hepatitis with seroreversion from anti-HBs to HBsAg after four weeks of alemtuzumab. Lamivudine (100 mg die) achieved a complete clinical recovery and HBV-DNA clearance from blood within 8 weeks. The second patient (HBsAg and HBV-DNA seronegative, anti-HBs and anti-HBc positive before treatment) was kept under prophylaxis with lamivudine up to three months after alemtuzumab. Two months after withdrawal of lamivudine, clinical and laboratory features of acute hepatitis B developed. Lamivudine therapy was restarted and obtained a prompt recovery with HBsAg and HBV-DNA clearance. These cases suggest that alemtuzumab, as all other immune response modifiers, should be used cautiously when HBV infection is present. While a recommendation for universal pre-emptive treatment with lamivudine of all HBsAg positive patients undergoing effective immunosuppressive treatment can be made it is more difficult to establish a pattern for patients who have serological markers of previous HBV infection (anti-HBs and/or anti-HBc) and even more for those with no HBV markers. Testing for HBV-DNA in serum by highly sensitive PCR may discover up to 50% of these cases, allowing preemptive lamivudine therapy. A substantial proportion would however go undiscovered in the absence of a liver biopsy, which is clearly an unfeasible proposition as a screening procedure. Once a reactivation has been documented, nucleoside analog therapy should last for a few months after HBV-DNA clearance.

Description: Introduction: Genetic modification of autologous hematopoietic stem and progenitor cells (HSPC) is a promising clinical intervention to cure inherited monogenic diseases. Successful gene therapy trials have already been conducted using CD34+ cells from bone marrow and from mobilized peripheral blood. In this regard, cord blood (CB) represents an attractive source of HSCs due to its high concentration of high proliferative HSPC and increased susceptibility to be transduced by lentiviral vectors. Unfortunately, the major disadvantage is the limited number of HSC in the CB collection. Consequently, ex-vivo expansion of CB-HSC is desirable to extend clinical applications. Purposes: To investigate the ability of UCB-cd34+ cells to be expanded in serum-free media supplemented with the early acting hematopoietic cytokines SCF,TPO and Flt-3 ligant (STF) and to characterize CD34+ cells subtypes, clonogenic capacity and gene expression profile during expansion. We also wanted to investigate the susceptibility of the expanded cd34+ cells to be transduced by a GFP-lentiviral vector (LV-GFP) Material and Methods: CD34+ immunoselected cells from 10 UCB were grown for 8 days in customized serum-free medium formulated for HSC expansion, supplemented with STF cytokines. Numbers end frequency of CD34+cells and co-expression of the primitive surface antigens (CD38, CD133, CD90) was evaluated during expansion. Colonies developed in methylcellulose were scored for enumeration ad typing. LV-GFP transduction efficiency was evaluated in CD34+ cells cultured for 4 days in expansion medium plus STF and for 24 hrs in X-vivo10 medium with STF±IL-3 cytokines; the last condition slightly expands CD34+ cells (1.3 fold) and are currently used for HSPC-lentivector transduction in gene therapy clinical trials. The transduction efficiency was evaluated by measuring the percentage of GFP+ cells in the bulk and in colonies developed in methylcellulose and the VCN/cell by Q-PCR. Gene expression profiles were analyzed by human whole genome Agilent microarray Technology to detect differentially expressed genes between expanded, ex-vivo medium cultured and un-cultured cells. Results: We found an average of 8 fold-increase CD34+cells at day 4 and of 22 fold- increase at day 8 of culture. The frequency of CD34+ was maintained at day 4 and declined of about 50% at day 8. CD34+/CD38- early progenitors doublet as early as day 4, differences in CD34+/CD133+ and CD34+/CD90+cells were not significant. The number of CFU slightly increased during expansion while the relative frequency of colonies type did not significantly changed. Four days expanded CD34+ cells were transduced more efficiently than those grown in ex-vivo medium even in presence of IL-3 added to the STF cytokine cocktail. Comprehensive gene expression profile analysis highlighted about 4000 genes differentially expressed in CD34+ cells expanded for 4 and for 8 days compared to that of the un-cultured cells. Conversely, the expression profiles analysis did not show any clear separation between different cell culture methods (expansion vs ex-vivo medium). Specifically, the number of differentially expressed genes in common between the different culture conditions compared with the un-cultured cells was statistically significant. Unsurprisingly, the common up-regulated genes were related to the cell cycle. The likeness between the gene expression profiles of the different culture conditions was also validated by the identification of a significantly small number of differentially expressed genes between them. Conclusions: UCB-CD34+ cells can be efficiently expanded and transduced in serum free conditions. The expanded cells exhibited phenotypic marchers typical of early progenitors and developed colonies in number and in type similar to the unmanipulated cells and exhibited whole gene expression profile that is consisted with that of CD34+ cells exposed for the short term culture conditions currently used in gene therapy trial mediated by lentiviral vectors. Results from this study open a window on the future possibility of using homologous UCB-HSC as target for gene correction in patients diagnosed for a genetic disorder in prenatal time. The genetically modified cells would be stored and used for gene therapy in the same individual in pediatric age. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F12000150004 Disclosures No relevant conflicts of interest to declare.

Description: Hematopoietic stem cell engineering is a promising therapy to cure b-thalassemia, in particular for patients who lack a suitable BM donor for allogeneic transplantation. Since the engrafted gene-corrected stem cells will not have any selective advantage over the unmodified ones, the effectiveness of the therapy in this setting largely depends on the infusion of high numbers of gene-modified cells and on the conditioning regimen. The quality of the infused cells is also crucial for the clinical outcome and the duration of the therapeutic effect. HSPCs mobilization, particularly when G-CSF and plerixafor are used in combination, has been proved to be the optimal approach to harvest a large number of CD34+cells in patients with hematological malignancies and in healthy volunteers. However adult heavily-transfused thalassemia patients have intrinsic characteristics that may adversely affect both the safety and the efficacy of mobilization. We conducted a clinical trial to investigate the safety and effectiveness of mobilizing HSPCs with G-CSF+plerixafor in adult patients affected by β-thalassemia major with the aim to reach a cell dose of ≥8x106 CD34+cells/Kg. We studied the kinetic of CD34+cells during mobilization and performed a comprehensive characterization of their molecular and functional properties. All patients completed the mobilization according to the protocol (G-CSF 10 μg/kg/day for four days, followed by plerixafor 240 μg/kg in the evening on day 4) and no serious adverse events occurred. Leukapheresis was done 10-12 hours after plerixafor (on day 5). Three of the four patients reached the target cell dose or more in single-apheresis collections, even one patient where a significant dose reduction of G-CSF was halved due to early hyperleukocytosis. For one patients the number of cells collected in the first apheresis was slightly below the established target and therefore, according to the protocol, she was subjected to a second apheresis on day 6, after an additional dose of plerixafor. The total yield from the combined apheresis in this patient was 13.0 CD34+cells /Kg. CD34+ cell yields per single apheresis in our patients were comparable to those in healthy donors (12 pts) mobilized in our hospital with G-CSF alone. A significant increase in the mean peripheral blood CD34+ cells (12.1± 8.2 fold), was unanimously observed after plerixafor addiction. The frequencies of the more primitive CD34+cell subtypes (CD34+CD38- and CD34+CD38-133+) as well as the clonogenic capacity tested in short term in vitro assay were found significantly increased too. Comprehensive microarray analysis of genes expressed in the CD34+ cells purified from the same patient upon mobilization with G-CSF alone (G/CD34+cell) and with G-CSF+plerixafor (G+pl/CD34+cell) highlighted a different HSCs repertoire. According to the mechanism of plerixafor mobilization, CXCR-4 gene expression was found 5-fold higher in G+pl/CD34+cells. CXCR-4 gene is known to be expressed on the surface of more primitive CD34+ HSCs with long-term repopulating potential and plays a central role in the regulation of adhesion of them to native niche in the BM. A substantial number of genes with previously shown implication in mechanisms of homing and engraftment (CXCR4, CD82, DPP4, ROBO4), or genes linked to stress resistance (CXCL4, SOD2, IL8, PPBP) as well as several chemokines genes involved in cell mobility (CXCL2, CXC3, CXCR2) were also found to be up-regulated in G+pl/CD34+cells. Overall, the yields, the primitive signatures of CD34+cells indicate the G-CSF+plerixafor mobilized peripheral blood as optimal graft that should favor HSPCs engraftment after transplantation. This findings has therapeutic implications not only for b-thalassemia but also for other hematopoietic stem cell gene therapy applications. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F1200015000. Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION Secondary Acute Myeloid Leukemia (s-AML) evolves from a previous hematopoietic clonal disease such as Myelodysplastic Syndromes (MDS/AML), myeloproliferative neoplasia (NPM/AML), medullary insufficiencies - aplastic anemia - (AA/AML) or exposure to chemo or radiotherapy (t-AML). The objective of this work is to describe and highlight the demographic, pathophysiologic and clinical-therapeutic characteristics of s-AML patients compared with p-AML. METHODS This is a retrospective cohort study based on the casuistry from 34 reference centers in Latin America during a period of 10 years (JAN10'-MAY19'). Patients ≥18 years old with primary AML, excluding the promyelocytic subtype (p-AML), and s-AML were admitted. Age, gender, performance status, comorbidity, cytogenetics, mutations, AML subtypes, extramedullary compromise, treatments and overall survival (OS) were analyzed. Statistically, Graph Pad Prism version 5.00 and, SPSS version 17 were used. RESULTS One thousand eleven patients with newly diagnosed AML were recruited, 693 (68.5%) corresponded to p-AML and 318 (31.5%) to s-AML. The demographic differences between p-AML and s-AML are shown on Table 1. Subtypes of s-AML: t-AML (18.5%), MDS/AML (58.2%), NMP/AML (13.5%), AA/AML (5.7%) and others s-AML (4.1%). Global median age was 58 years (R 18-93) and male 52%. Extramedullary compromise in CNS (3.2%) and other organs (5.5%). Seven hundred ninety-three cytogenetic studies were evaluable (based on MRC classification): High (22.3%), Intermediate (68.3%) and Low Risk (9.3%). FLT3 mutation was more frequently found in p-AML. In s-AML, the multivariate study showed short overall survival associated with ECOG ≥2 (HR:2.0), white blood count ≥ 50x109/L at diagnosis (HR:1.9), poor risk karyotype (HR:1.6) and age over 60 years (HR:1.5). At least, 883 patients received treatment (Table 2). During this study period, 211 patients (21%) were transplanted; 49 (23%) were s-AML; histoidentical related donor (46%), haploidentical (39%), non-related (8%) and autologous (7%). The median survival for all AML was 11.0 months with a statistically significant difference in favor of the p-AML (Figure 1). CONCLUSION Performance status (by ECOG ≥2), age ≥60, level of leukocytes a ≥50x109/L, poor risk karyotype and s-AML subtype at diagnosis had a significant worse impact on overall survival. Most patients with s-AML came from MDS, they were older and their incidence increased as the population aged. They presented more comorbidities and worse performance status. Undoubtedly, our findings showed that s-AML is a distinct high risk subset of myeloid disorder with adverse prognosis and represents a therapeutic challenge. Disclosures No relevant conflicts of interest to declare.

Description: The Bcl-2 protein is capable of preventing apoptosis, and in vitro evidence suggests a role in drug resistance. It is expressed and the gene is rearranged in a proportion of cases of large-cell non-Hodgkin's lymphoma (NHL), but the clinical significance of these findings is controversial. The purpose of this study was to determine the influence of both Bcl-2 expression and major breakpoint region (MBR) bcl-2 rearrangement in a large cohort of prospectively accrued patients with intermediate-grade B-cell NHL treated in a standardized manner. All patients with Working Formulation F, G, or H NHL treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy in British National Lymphoma investigation studies between July 1974 and April 1992 were considered for this study if the appropriate paraffin blocks were available. Paraffin sections from the diagnostic specimen were analyzed for evidence of MBR rearrangement using a polymerase chain reaction-based method, and for Bcl-2 expression using immunohistochemistry. Failure to achieve complete remission (CR), relapse, death from NHL, and deaths from all causes were used as end points to measure CR rate, actuarial relapse rate, actuarial survival from NHL, and actuarial overall survival. One hundred sixty-one suitable patients were identified and tested for the bcl-2 MBR translocation, with 27 (17%) found to be positive; 153 of these patients were tested with immunocytochemistry, and 84 (55%) showed evidence of Bcl-2 expression. For patients who achieved CR from the initial treatment, the relapse rate was significantly higher in those with Bcl-2 expression than in those without. In addition, multivariate analysis identified Bcl-2 expression as the only factor significantly related to relapse rate in the subjects measured. The cause-specific survival for NHL in the series as a whole was significantly lower in patients with Bcl-2 expression than in those without. MBR status had no significant influence on any of the outcome measures, but the number of MBR-positive patients was relatively small, and larger studies are required. In conclusion, in Working Formulation F, G, and H NHL of B-cell type, expression of Bcl-2 protein predicted independently for relapse.