A phase II trial of lenalidomide plus rituximab in previously untreated follicular non-Hodgkin's lymphoma (NHL): CALGB 50803 (Alliance).

BACKGROUND: This multicenter, phase II trial tested the tolerability and efficacy of lenalidomide plus rituximab in patients with previously untreated follicular lymphoma (FL). PATIENTS AND METHODS: Patients with grade 1-3a FL, stage 3-4 or bulky stage 2, FL international prognostic index (FLIPI) 0-2, and no prior therapy were eligible to receive rituximab 375 mg/m2 weekly during cycle 1 and day 1 of cycles 4, 6, 8, and 10, plus lenalidomide 20-25 mg on days 1-21 for twelve 28-day cycles. The primary objectives were to evaluate response rates [complete (CR) and overall] and time to progression. Secondary objectives included toxicity, response according to polymorphisms in FcgR2A and FcgR3A, and changes in circulating pro-angiogenic cells. RESULTS: From October 2010 to September 2011, 66 patients were enrolled. Median age was 53 years, 34 were female, 15 had bulky disease, 21 were FLIPI 0-1, 43 FLIPI 2, and 2 FLIPI 3. One patient withdrew before receiving treatment. Fifty-one patients completed 12 cycles of lenalidomide. Reasons for discontinuation included withdrawal (n = 6), adverse events (n = 6), progression (n = 2). Grade 3-4 hematologic toxicity included neutropenia (21%), lymphopenia (9%), and thrombocytopenia (2%), infection (11%), and rash (8%). Grade 1-2 toxicity included fatigue (78%), diarrhea (37%), rash (32%), and febrile neutropenia in one patient. The overall response rate was 95%; the CR rate was 72% (95% confidence interval, 60% to 83%). With a median follow-up of 5 years, the 2- and 5-year progression-free survival were 86% and 70%, respectively, and the 5-year overall survival was 100%. There was no association between CR rate or PFS and FLIPI, histological grade, bulky disease, FcgR2A/FcgR3A polymorphism, or change in circulating endothelial cell/hematopoietic progenitor cell. CONCLUSION: Lenalidomide plus rituximab was associated with low rates of grade 3-4 toxicity, yielded a CR rate and PFS similar to chemotherapy-based treatment and may represent a reasonable alternative to immunochemotherapy in previously untreated FL. CLINICALTRIALS.GOV IDENTIFIER: NCT01145495.

International Working Group consensus response evaluation criteria in lymphoma (RECIL 2017).

In recent years, the number of approved and investigational agents that can be safely administered for the treatment of lymphoma patients for a prolonged period of time has substantially increased. Many of these novel agents are evaluated in early-phase clinical trials in patients with a wide range of malignancies, including solid tumors and lymphoma. Furthermore, with the advances in genome sequencing, new "basket" clinical trial designs have emerged that select patients based on the presence of specific genetic alterations across different types of solid tumors and lymphoma. The standard response criteria currently in use for lymphoma are the Lugano Criteria which are based on [18F]2-fluoro-2-deoxy-D-glucose positron emission tomography or bidimensional tumor measurements on computerized tomography scans. These differ from the RECIST criteria used in solid tumors, which use unidimensional measurements. The RECIL group hypothesized that single-dimension measurement could be used to assess response to therapy in lymphoma patients, producing results similar to the standard criteria. We tested this hypothesis by analyzing 47 828 imaging measurements from 2983 individual adult and pediatric lymphoma patients enrolled on 10 multicenter clinical trials and developed new lymphoma response criteria (RECIL 2017). We demonstrate that assessment of tumor burden in lymphoma clinical trials can use the sum of longest diameters of a maximum of three target lesions. Furthermore, we introduced a new provisional category of a minor response. We also clarified response assessment in patients receiving novel immune therapy and targeted agents that generate unique imaging situations.

Toxicity burden score: a novel approach to summarize multiple toxic effects.

BACKGROUND: Toxicity data from cancer trials are summarized into a single outcome, dose-limiting toxicity (DLT), which does not account for multiple lower grade toxic effects nor differentiates between toxicity types and gradations within DLT. METHODS: Toxicity data were summarized into a toxicity burden score (TBS) using a weighted sum. The severity weights were estimated via regression using historical data. We demonstrated the method using historical data from a bortezomib trial and illustrated the advantages of defining DLT based on TBS in a simulated dose-finding trial. RESULTS: The estimated weights were 0.17, 0.40 and 0.85 for grade 1/2, grade 3 and grade 4 platelets, respectively; 0.19, 0.64, 1.03 and 2.53 for grade 1, 2, 3 and 4 neuropathy, respectively and 0.17 for each grade 3 or higher nonhematologic toxic effects unrelated to treatment. In the simulated trial, the probability of selecting doses above the maximum tolerated dose decreased when using the DLT defined based on TBS. CONCLUSIONS: TBS is a feasible approach to summarize toxicity. It includes information from the grades and types of multiple toxic effects and can be applied in all phases of drug development. Further efforts should focus on validating the method in a large prospective study before applying it in practice.

Phase II trial of galiximab (anti-CD80 monoclonal antibody) plus rituximab (CALGB 50402): Follicular Lymphoma International Prognostic Index (FLIPI) score is predictive of upfront immunotherapy responsiveness.

BACKGROUND: This phase II CALGB trial evaluated the activity and safety of an extended induction schedule of galiximab (G) plus rituximab (R) in untreated follicular lymphoma (FL). PATIENTS AND METHODS: Patients with previously untreated FL (grades 1, 2, 3a) received 4 weekly infusions of G + R, followed by an additional dose every 2 months four times. International Workshop Response Criteria were used to evaluate response. RESULTS: Sixty-one patients were treated and antibody infusions were well tolerated. The overall response rate (ORR) is 72.1% (95% confidence interval 59.2% to 82.9%): 47.6% complete response (CR)/unconfirmed complete response (CRu) and 24.6% partial response. At a median follow-up time of 4.3 years (range, 0.3-5.3 years) median progression-free survival (PFS) is 2.9 years. Notably, Follicular Lymphoma International Prognostic Index (FLIPI) correlated with ORR, CR rate, and PFS, and the low-risk FLIPI group (n = 12) achieved a 92% ORR, 75% CR/CRu rate, and 75% 3-year PFS. CONCLUSIONS: An extended induction schedule of G + R in previously untreated FL is well tolerated and appears particularly efficacious in those patients with low-risk FLIPI scores. In addition, this trial served as the initial platform for additional CALGB 'doublet' combination regimes of rituximab plus other novel targeted agents.

Prevention of experimental autoimmune encephalomyelitis by antibodies against interleukin 12.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system that can be transferred to naive mice via CD4+ T cells isolated from appropriately immunized mice. We have evaluated the effects of recombinant murine interleukin 12 (rmIL-12), a potent inducer of interferon gamma (IFN-gamma) and promoter of Th1 T cell development, on the course of adoptively transferred EAE. The transfer of lymph node cells (LNC) isolated from proteolipid protein (PLP)-primed animals and stimulated in vitro with PLP to naive mice resulted in a progressive paralytic disease culminating in complete hind limb paralysis in the majority of the recipients. When mice were injected with LNC that had been stimulated in vitro with PLP in the presence of rmIL-12, the subsequent course of disease was more severe and prolonged. The addition of rmIL-12 during the in vitro stimulation with PLP resulted in a 10-fold increase in IFN-gamma and a 2-fold increase in tumor necrosis factor (TNF) alpha in the supernatants, relative to LNC stimulated with PLP alone. However, neutralization of IFN-gamma or TNF-alpha in vitro with specific antibodies did not abrogate the ability of rmIL-12 to exacerbate the subsequent disease. Similarly, mice treated with rmIL-12 in vivo after the transfer of antigen-stimulated LNC developed a more severe and prolonged course of disease compared with vehicle-treated control animals. In contrast, treatment of mice with an antibody to murine IL-12 after cell transfer completely prevented paralysis, with only 40% of the mice developing mild disease. These results demonstrate that in vitro stimulation of antigen primed LNC with PLP and rmIL-12 enhances their subsequent encephalitogenicity. Furthermore, inhibition of endogenous IL-12 in vivo after LNC transfer prevented paralysis, suggesting that endogenous IL-12 plays a pivotal role in the pathogenesis of this model of autoimmune disease.

Surveillance CT scans are a source of anxiety and fear of recurrence in long-term lymphoma survivors.

BACKGROUND: We aimed to assess anxiety and the psychological impact of routine surveillance scans in long-term survivors of adult aggressive lymphoma. PATIENTS AND METHODS: In this cross-sectional observational study of 70 survivors of curable adult aggressive lymphoma, we measured anxiety and the doctor-patient relationship and performed a qualitative interview (n = 30) focused on patient perception of routine follow-up imaging studies. RESULTS: Participants were diagnosed with aggressive lymphoma a median of 4.9 years (2.4-38.0 years) before enrollment. Thirty-seven percent of patients were found to meet criteria for clinically significant anxiety, which was not associated with years since diagnosis. In multivariate analysis, history of relapse and a worse doctor-patient relationship were independently associated with higher anxiety levels. Despite representing a largely cured population, in qualitative interviews patients reported fear of recurrence as a major concern and considerable anxiety around the time of a follow-up imaging scan. CONCLUSIONS: Routine surveillance scans exacerbate underlying anxiety symptoms and fear of recurrence in survivors of aggressive lymphoma. Strategies to minimize follow-up imaging and to improve doctor-patient communication should be prospectively evaluated to address these clinically significant issues.

Angiogenesis and antiangiogenic therapy in non-Hodgkin's lymphoma.

Angiogenesis, the growth of new blood vessels, requires dynamic expansion, assembly and stabilization of vascular endothelial cells in response to proangiogenic stimuli. Antiangiogenic strategies have become an important therapeutic modality for solid tumors. While many aspects of postnatal pathological angiogenesis have been extensively studied in the context of nonhematopoietic neoplasms, the precise role of these processes in lymphoma pathogenesis is under active investigation. Lymphoma growth and progression is potentiated by at least two distinct angiogenic mechanisms: autocrine stimulation of tumor cells via expression of vascular endothelial growth factor (VEGF) and VEGF receptors by lymphoma cells, as well as paracrine influences of proangiogenic tumor microenvironment on both local neovascular transformation and recruitment of circulating bone marrow-derived progenitors. Lymphoma-associated infiltrating host cells including hematopoietic monocytes, T cells and mesenchymal pericytes have increasingly been associated with the pathogenesis and prognosis of lymphoma, in part providing perivascular guidance and support to neoangiogenesis. Collectively, these distinct angiogenic mechanisms appear to be important therapeutic targets in selected non-Hodgkin's lymphoma (NHL) subtypes. Understanding these pathways has led to the introduction of antiangiogenic treatment strategies into the clinic where they are currently under assessment in several ongoing studies of NHL patients.

Bortezomib in patients with relapsed or refractory mantle cell lymphoma: updated time-to-event analyses of the multicenter phase 2 PINNACLE study.

BACKGROUND: We previously reported results of the phase 2, multicenter PINNACLE study, which confirmed the substantial single-agent activity of bortezomib in patients with relapsed or refractory mantle cell lymphoma (MCL). MATERIALS AND METHODS: We report updated time-to-event data, in all patients and by response to treatment, after extended follow-up (median 26.4 months). RESULTS: Median time to progression (TTP) was 6.7 months. Median time to next therapy (TTNT) was 7.4 months. Median overall survival (OS) was 23.5 months. In responding patients, median TTP was 12.4 months, median duration of response (DOR) was 9.2 months, median TTNT was 14.3 months, and median OS was 35.4 months. Patients achieving complete response had heterogeneous disease characteristics; among these patients, median TTP and DOR were not reached, and median OS was 36.0 months. One-year survival rate was 69% overall and 91% in responding patients. Median OS from diagnosis was 61.1 months, after median follow-up of 63.7 months. Activity was seen in patients with refractory disease and patients relapsing following high-intensity treatment. Toxicity was generally manageable. CONCLUSIONS: Single-agent bortezomib is associated with lengthy responses and notable survival in patients with relapsed or refractory MCL, with considerable TTP and TTNT in responding patients, suggesting substantial clinical benefit.

Agonist-induced myopathy at the neuromuscular junction is mediated by calcium.

Inactivation of cholinesterases at mammalian neuromuscular junctions (nmj) produces extensive muscle "necrosis." Fine-structurally, this myopathy begins near the nmj with an increase in large-diameter vesicles in the soleplasm, the dissolution of Z-disks, dilation of mitochondria, destruction of sarcoplasmic reticulum, and often a highly specific contracture of the muscle under the endplate. Since a Ca++-activated protease which specifically removes Z-disks is known to exist in mammalian skeletal muscle, we tested the possibility that the myopathy after esterase inactivation is due to the prolongation of acetylcholine lifetime and thus of Ca++ influx. We first produced the myopathy near endplates by inactivating esterases with diisopropylfluorophosphate (DFP) followed by nerve stimulation for 1--2 h in vitro. The myopathy was later mimicked by bath application of carbamylcholine without esterase inhibitors. This myopathy could be prevented by inactivating the acetylcholine receptors (AChR) with alpha-bungarotoxin (alpha-BGT) or by removing Ca++ from the bath with EGTA. These results favor the hypothesis that esterase inhibition leads to an agonist-induced myopathy, which is mediated by Ca++ and requires an intact AChR.

Preclinical and clinical evaluation of epratuzumab (anti-CD22 IgG) in B-cell malignancies.

The vast majority of non-Hodgkin's lymphomas are of B-cell phenotype. Development of unlabeled or radiolabeled therapeutic monoclonal antibodies against the cell surface antigen, CD20, has revolutionized the treatment of these malignancies. It is clear that antibodies targeting other B-cell-specific molecules, such as CD22, also offer potential therapeutic benefit. Epratuzumab is a humanized anti-CD22 monoclonal, which has undergone preclinical and phase I/II clinical evaluation in patients with indolent or aggressive lymphoma. Data suggest that this agent is well tolerated, and can induce tumor regressions. Trials are currently evaluating its safety and activity in combination with rituximab (chimeric anti-CD20) and standard chemotherapy are ongoing. Initial results suggest that these regimens have acceptable toxicity, and that epratuzumab warrants further evaluation as an adjunct to standard lymphoma treatment regimens.

Apparent desensitization of NMDA responses in Xenopus oocytes involves calcium-dependent chloride current.

N-Methyl-D-aspartate (NMDA) receptors were expressed and studied in Xenopus oocytes injected with rat brain RNA. NMDA application elicits a rapid inward current that decays in several seconds to a relatively stable level. This decay is reportedly due to desensitization. However, we found the early transient component could be evoked more than once during a single application of NMDA, suggesting that the receptor did not actually desensitize. Removal of external Ca2+, replacement of Ca2+ with Ba2+, or intracellular injection of EGTA abolished the transient component. Furthermore, a variety of Cl- channel blockers nearly eliminated the transient component and inhibited the plateau current as well. We propose that a significant portion of the NMDA current recorded in oocytes is carried by a transient inward Cl- current triggered by Ca2+ influx through the NMDA receptor/channel.

Distinct calcium channels are generated by alternative splicing and are differentially expressed in the mammalian CNS.

A number of pharmacologically and electrophysiologically distinct voltage-dependent Ca2+ channels have been identified in mammalian neurons. Two rat brain Ca2+ channel alpha 1 subunits (rbC-I and rbC-II) have been isolated by molecular cloning and shown to be highly related (95%) to the cardiac dihydropyridine-sensitive Ca2+ channel. The rbC-II protein is distinct from rbC-I in that it contains a 3 amino acid insert in the putative cytoplasmic loop between domains II and III and a 28 amino acid substitution corresponding to the third transmembrane segment (S3) of the fourth domain. We show that rbC-I and rbC-II transcripts are generated by alternative splicing and that they are differentially expressed in the rat CNS.

Combined PET and low-dose, noncontrast CT scanning obviates the need for additional diagnostic contrast-enhanced CT scans in patients undergoing staging or restaging for lymphoma.

BACKGROUND: Positron emission tomography (PET) is more accurate than computed tomography (CT) in staging and restaging of lymphoma, but both are considered necessary. Increasingly, PET is carried out with a low-dose CT scan. Many patients undergo both PET/CT and standard diagnostic CT. The clinical utility of performing both studies in patients with lymphoma was evaluated. PATIENTS AND METHODS: Patients with lymphoma who underwent concurrent PET/CT and diagnostic CT (a scan pair) were identified, and findings detected in either scan but not both were documented. Discrepancies were considered significant if they were related to either lymphoma or another disease process which potentially required intervention. RESULTS: Eighty-seven scan pairs were identified. PET/CT detected additional lesions over diagnostic CT in 30 patients, of which 11 demonstrated increased clinical stage. Lymphoma therapy changed based on PET/CT in two patients, and one occult rectal cancer was detected. In contrast, diagnostic CT detected five relevant findings, including two incidental findings (venous thrombosis) and three patients with splenic lesions, none of which could be confirmed as lymphoma. No patient had change of stage or lymphoma therapy based on diagnostic CT. CONCLUSION: In our series, diagnostic CT did not add value to staging or restaging of lymphoma when carried out concurrently with PET/CT.

Intensive treatment strategies may not provide superior outcomes in mantle cell lymphoma: overall survival exceeding 7 years with standard therapies.

BACKGROUND: Reported median overall survival (OS) in patients with mantle cell lymphoma (MCL) has been reported to be just 3-4 years. As a consequence, first-line treatment has become more aggressive. Single-center studies with R-Hyper-CVAD and/or autologous stem-cell transplant (ASCT) have produced 3-year OS rates >80%, prompting many to adopt their use. We evaluated outcomes from a single-center cohort managed in a more traditional fashion. METHODS: We identified patients with MCL evaluated at Weill Cornell Medical Center since 1997, and included those with known date of diagnosis. An online social security database was used to verify survival. RESULTS: We identified 181 patients with MCL, and date of diagnosis could be determined in 111. Three-year OS from diagnosis was 86% [95% confidence interval (CI) 78% to 92%]. Median OS was 7.1 years (95% CI 63-98 months). Adequate information on therapy was available for 75 patients. Only five were treated upfront with (R)-Hyper-CVAD or ASCT while an additional four patients received one of these regimens subsequently. Treatment type had no significant effect on OS. CONCLUSION: Outcomes with standard approaches can yield similar survival to that achieved with more intensive approaches. Biases may account for the perceived superiority of aggressive strategies.

Stability of zinc oxide nanofluids prepared with aggregated nanocrystalline powders.

Aqueous zinc oxide (ZnO) suspensions were prepared using a two-step preparation method in which an aggregated nanocrystalline ZnO powder was dispersed in water using a polyelectrolyte. The fluid showed anomalously high thermal conductivity when compared with the Maxwell and Hamilton-Crosser predictions. However, analysis of the particle size distribution showed that the fluid contained aggregated 20 nm crystallites of ZnO with a high volume fraction of particles larger than 100 nm. Sedimentation experiments revealed that particles settled out of the stationary fluid over times ranging from 0.1 hours to well over 10,000 hours. The size of the particles remaining in suspension agreed well with predictions made using Stoke's law, suggesting flocculation was not occurring in the fluids. Finally, a new concept of nanofluid stability is introduced based on the height of the fluid, sedimentation, Brownian motion and the kinetic energy of the particles.

Regulation of the inflammatory response in animal models of multiple sclerosis by interleukin-12.

Interleukin 12 (IL-12), a novel heterodimeric protein produced primarily by antigen-presenting cells, serves as a key regulator of innate and adaptive immune responses. In addition to being a potent inducer of IFN-gamma, IL-12 is widely considered to be the principal cytokine that regulates the generation of Th1 type effector cells. As the successful induction of experimental autoimmune encephalomyelitis (EAE) is associated with a strong Th1 type cellular response, we have evaluated the role of IL-12 in regulating the pathogenesis of EAE in SJL/J mice and Lewis rats. In both settings, treatment with IL-12 was found to accelerate the onset and increase the severity and duration of clinical disease. More importantly, administration of IL-12 to Lewis rats that had recovered from primary disease was found to trigger clinical relapse. In all instances, IL-12-induced exacerbation was associated with a profound increase in iNOS positive macrophages within the perivascular lesions. Although IL-12-induced IFN-gamma does not appear to be required for exacerbation of disease, neutralizing antibodies against murine IL-12 delay the onset and reduce the severity of adoptively transferred EAE, indicating a role for endogenous IL-12 as regulator of disease. Based on the above findings, effective inhibition of IL-12 in vivo may have great therapeutic value in the treatment of MS and other Th1-associated inflammatory disorders.

Elitek-rasburicase: an effective means to prevent and treat hyperuricemia associated with tumor lysis syndrome, a Meeting Report, Dallas, Texas, January 2002.

Renal precipitation of uric acid associated with tumor lysis syndrome (TLS) is a major complication in the management of leukemia, lymphoma, and other drug-sensitive cancers. Management of hyperuricema has historically consisted of administration of allopurinol, hydration, alkalinization to maintain pH between 7.0 and 7.3, and in some cases diuresis. Allopurinol, a xanthine analogue, blocks xanthine oxidase and formation of uric acid. Urate oxidase converts uric acid to allantoin, which is 5-10 times more soluble than uric acid. Homo sapiens cannot express urate oxidase because of a nonsense mutation. Urate oxidase was initially purified from Aspergillus flavus fungus. Treatment with this nonrecombinant product had been effective in preventing renal precipitation of uric acid in cancer patients, but was associated with a relatively high frequency of allergic reactions. This enzyme was recently cloned from A. flavus and is now manufactured as a recombinant protein. Clinical trials have shown this drug to be more effective than allopurinol for prevention and treatment of hyperuricemia in leukemia and lymphoma patients. This drug has been approved in Europe as well as the US and several clinical trials are in progress to further determine its clinical utility in other patient subsets. The purpose of this meeting was to discuss usefulness of recombinant urate oxidase, also known as rasburicase, Fasturtec, and Elitek, for the management of TLS in certain cancer patients.

Constant subcutaneous infusion of rhIL-11 in mice: efficient delivery enhances biological activity.

Previous studies have shown that subcutaneous (SC) bolus administration of recombinant human interleukin-11 (rhIL-11) stimulates megakaryocytopoiesis and increases peripheral platelet counts in naive mice. This study was designed to determine whether administration of rhIL-11 by constant SC infusion altered either the magnitude of the nature of the hematologic response. Female C57BL/6 mice were implanted subcutaneously with 7-day Alzet mini-osmotic pumps containing either rhIL-11 with 0.5% homologous mouse serum (delivery rate of 250 microg/kg/d) or vehicle alone. Mice were sacrificed on days 3, 7, 10, and 13 after pump implantation, and the hematopoietic response was compared to mice receiving an equivalent dose of rhIL-11 administered by SC injection (250 microg/kg/d, 7 days) or vehicle controls. Subcutaneous injection of rhIL-11 resulted in a significant increase in peripheral platelet counts with a maximum platelet increase of 44% over controls observed on day 7 of the study. Platelet counts subsequently declined (24% by day 10) returning to control values by day 13. The increase in peripheral platelet counts was accompanied by an increase in reticulated platelets on day 7 and a shift to higher ploidy bone marrow megakaryocytes on days 3 and 7. Compared to SC injection, both the magnitude and duration of the platelet increase were significantly enhanced following continuous SC infusion of rhIL-11. Maximum platelet counts were detected on day 10 (115% above vehicle controls), and platelets remained significantly elevated on day 13 (84%), 6 days after rhIL-11 administration had stopped. Consistent with the platelet response, the modal ploidy of bone marrow megakaryocytes was shifted from 16N to 32N on days 3 and 7, with increases in 32N megakaryocytes still apparent on days 10 and 13. There was also a significant increase in reticulated platelets detected in the peripheral blood on days 3, 7, and 10 compared to mice administered rhIL-11 by SC injection, The changes in reticulated platelets and bone marrow megakaryocyte ploidy are consistent with the increased and prolonged platelet response following SC infusion of rhIL-11. In addition to the effects observed on peripheral platelet counts, constant SC infusion of rhIL-11 dramatically enhanced splenic hematopoietic activity, increasing spleen weight and cellularity as well as splenic megakaryocyte, erythroid, granulocyte, and macrophage progenitors compared to mice receiving rhIL-11 by SC injection.

Retroviral transduction of human CD34+ umbilical cord blood progenitor cells with a mutated dihydrofolate reductase cDNA.

Umbilical cord blood cells (UCB) have become a major target population for experimental and clinical studies using transfer of genes involved in inborn enzymatic diseases. Cord blood contains hematopoietic progenitor cells at a high frequency, and expanding these cells ex vivo generates sufficient numbers of hematopoietic precursors for transplantation into adults, e.g., as supportive treatment. As clinical reports about retroviral transduction into UCB cells have not been as encouraging as the first preclinical data, we have established a retroviral transduction system that allows expansion and selection of hematopoietic progenitor cells from UCB. CD34-enriched UCB cells were transduced with a retroviral vector encoding a mutated dihydrofolate reductase cDNA that confers MTX resistance. We observed increased resistance to MTX in transduced granulocyte macrophage-colony forming units (CFU-GM) after co-culture of CD34+ UCB cells with the virus-producing cell line, or after incubation with virus-containing supernatant. The supernatant-based transduction protocol included a prestimulation with recombinant interleukin-1 (rhIL-1), rhkit-ligand, and rhIL-3 to increase the percentage of cells in S phase to greater than 50%. Using this protocol we measured a 72-fold expansion of CFU-GM and a 2.5-fold selective advantage of transduced versus nontransduced progenitor cells after exposure to low-dose methotrexate in liquid culture. Polymerase chain reaction analysis revealed integration of proviral DNA into the majority of transduced colonies before and after ex vivo expansion. The retroviral vector and transduction protocol reported here provides an experimental system for selection and expansion of retrovirally transduced progenitor/stem cells from UCB that may help improve the efficiency of current clinical gene therapy strategies.