Dabrafenib plus trametinib versus dabrafenib monotherapy in patients with metastatic BRAF V600E/K-mutant melanoma: long-term survival and safety analysis of a phase 3 study.

BACKGROUND: Previous analysis of COMBI-d (NCT01584648) demonstrated improved progression-free survival (PFS) and overall survival (OS) with combination dabrafenib and trametinib versus dabrafenib monotherapy in BRAF V600E/K-mutant metastatic melanoma. This study was continued to assess 3-year landmark efficacy and safety after ≥36-month follow-up for all living patients. PATIENTS AND METHODS: This double-blind, phase 3 study enrolled previously untreated patients with BRAF V600E/K-mutant unresectable stage IIIC or stage IV melanoma. Patients were randomized to receive dabrafenib (150 mg twice daily) plus trametinib (2 mg once daily) or dabrafenib plus placebo. The primary endpoint was PFS; secondary endpoints were OS, overall response, duration of response, safety, and pharmacokinetics. RESULTS: Between 4 May and 30 November 2012, a total of 423 of 947 screened patients were randomly assigned to receive dabrafenib plus trametinib (n = 211) or dabrafenib monotherapy (n = 212). At data cut-off (15 February 2016), outcomes remained superior with the combination: 3-year PFS was 22% with dabrafenib plus trametinib versus 12% with monotherapy, and 3-year OS was 44% versus 32%, respectively. Twenty-five patients receiving monotherapy crossed over to combination therapy, with continued follow-up under the monotherapy arm (per intent-to-treat principle). Of combination-arm patients alive at 3 years, 58% remained on dabrafenib plus trametinib. Three-year OS with the combination reached 62% in the most favourable subgroup (normal lactate dehydrogenase and <3 organ sites with metastasis) versus only 25% in the unfavourable subgroup (elevated lactate dehydrogenase). The dabrafenib plus trametinib safety profile was consistent with previous clinical trial observations, and no new safety signals were detected with long-term use. CONCLUSIONS: These data demonstrate that durable (≥3 years) survival is achievable with dabrafenib plus trametinib in patients with BRAF V600-mutant metastatic melanoma and support long-term first-line use of the combination in this setting.

Activity of single-agent melphalan 220-300 mg/m2 with amifostine cytoprotection and autologous hematopoietic stem cell support in non-Hodgkin and Hodgkin lymphoma.

High-dose chemotherapy using melphalan (HDMEL) is an important component of many conditioning regimens that are given before autologous hematopoietic stem cell transplantation (AHSCT). In contrast to the situation in myeloma, and to a lesser degree acute leukemia, only a very limited published experience exists with the use of HDMEL conditioning as a single agent in doses requiring AHSCT for lymphoma, both Hodgkin lymphoma (HL) and especially non-Hodgkin lymphoma (NHL). Thus, we report results of treating 26 lymphoma patients (22 with NHL and four with HL) with HDMEL 220-300 mg/m(2) plus amifostine (AF) cytoprotection and AHSCT as part of a phase I-II trial. Median age was 51 years (range 24-62 years); NHL histology was varied, but was aggressive (including transformed from indolent) in 19 patients, indolent in two patients and mantle cell in one. All 26 patients had been extensively treated; 11 were refractory to the immediate prior therapy on protocol entry and two had undergone prior AHSCT. All were deemed ineligible for other, 'first-line' AHSCT regimens. Of these 26 patients, 22 survived to initial tumor evaluation on D +100. At this time, 13 were in complete remission, including four patients who were in second CR before HDMEL+AF+AHSCT. Responses occurred at all HDMEL doses. Currently, seven patients are alive, including five without progression, with a median follow-up in these latter patients of D +1163 (range D +824 to D +1630); one of these patients had a nonmyeloablative allograft as consolidation on D +106. Conversely, 14 patients relapsed or progressed, including five who had previously achieved CR with the AHSCT procedure. Two patients, both with HL, remain alive after progression; one is in CR following salvage radiotherapy. Six patients died due to nonrelapse causes, including two NHL patients who died while in CR. We conclude that HDMEL+AF+AHSCT has significant single-agent activity in relapsed or refractory NHL and HL. This experience may be used as a starting point for subsequent dose escalation of HDMEL (probably with AF) in established combination regimens.

Acute renal failure requiring dialysis after allogeneic blood and marrow transplantation identifies very poor prognosis patients.

We examined the incidence, risk factors and associated mortality of acute renal failure requiring dialysis (Renal Bearman Grade [BG] 3) in a 3-year cohort of 97 consecutive allogeneic blood and marrow transplantation (alloBMT) patients. In all, 20 (21%) developed Renal BG3 (all died by day +132) and 77 (79%) developed renal insufficiency (Renal BG1-2). Renal BG3 was a contributing or primary cause of death in 18 (90%) patients who continued to require dialysis at time of death. The two Renal BG3 patients whose deaths were not related to renal failure died on day +103 of hemorrhage and day +132 of underlying disease. By univariate analysis, age, unrelated donor, veno-occlusive disease (VOD) and grade III-IV acute graft-versus-host disease with hepatic involvement were significantly associated with Renal BG3. The multivariate model of time to Renal BG3 determined only a prior diagnosis of severe acute GVHD (RR=4.1, 95% CI 1.6-10.3, P=0.003) and VOD (RR=9.1, 95% CI 3.5-23.7, P<0.001) as significant independent predictors. Renal BG3 is generally considered a conditioning regimen-related toxicity. This study demonstrates that Renal BG3 is most commonly a complication of hepatic co-morbidities after allogeneic blood and marrow transplantation and identifies patients with a very poor prognosis.

Autotransplantation for advanced lymphoma and Hodgkin's disease followed by post-transplant rituxan/GM-CSF or radiotherapy and consolidation chemotherapy.

Disease relapse occurs in 50% or more of patients who are autografted for relapsed or refractory lymphoma (NHL) or Hodgkin's disease (HD). The administration of non-cross-resistant therapies during the post-transplant phase could possibly control residual disease and delay or prevent its progression. To test this approach, 55 patients with relapsed/refractory or high-risk NHL or relapsed/refractory HD were enrolled in the following protocol: stem cell mobilization: cyclophosphamide (4.5 g/m(2)) + etoposide (2.0 g/m(2)) followed by GM-CSF or G-CSF; high-dose therapy: gemcitabine (1.0 g/m(2)) on day -5, BCNU (300 mg/m(2)) + gemcitabine (1.0 g/m(2)) on day -2, melphalan (140 mg/m(2)) on day -1, blood stem cell infusion on day 0; post-transplant immunotherapy (B cell NHL): rituxan (375 mg/m(2)) weekly for 4 weeks + GM-CSF (250 microg thrice weekly) (weeks 4-8); post-transplant involved-field radiotherapy (HD): 30-40 Gy to pre-transplant areas of disease (weeks 4-8); post-transplant consolidation chemotherapy (all patients): dexamethasone (40 mg daily)/cyclophosphamide (300 mg/m(2)/day)/etoposide (30 mg/m(2)/day)/cisplatin (15 mg/m(2)/day) by continuous intravenous infusion for 4 days + gemcitabine (1.0 g/m(2), day 3) (months 3 + 9) alternating with dexamethasone/paclitaxel (135 mg/m(2))/cisplatin (75 mg/m(2)) (months 6 + 12). Of the 33 patients with B cell lymphoma, 14 had primary refractory disease (42%), 12 had relapsed disease (36%) and seven had high-risk disease in first CR (21%). For the entire group, the 2-year Kaplan-Meier event-free survival (EFS) and overall survival (OS) were 30% and 35%, respectively, while six of 33 patients (18%) died before day 100 from transplant-related complications. The rituxan/GM-CSF phase was well-tolerated by the 26 patients who were treated and led to radiographic responses in seven patients; an eighth patient with a blastic variant of mantle-cell lymphoma had clearance of marrow involvement after rituxan/GM-CSF. Of the 22 patients with relapsed/refractory HD (21 patients) or high-risk T cell lymphoblastic lymphoma (one patient), the 2-year Kaplan-Meier EFS and OS were 70% and 85%, respectively, while two of 22 patients (9%) died before day 100 from transplant-related complications. Eight patients received involved field radiation and seven had radiographic responses within the treatment fields. A total of 72 courses of post-transplant consolidation chemotherapy were administered to 26 of the 55 total patients. Transient grade 3-4 myelosuppression was common and one patient died from neutropenic sepsis, but no patients required an infusion of backup stem cells. After adjustment for known prognostic factors, the EFS for the cohort of HD patients was significantly better than the EFS for an historical cohort of HD patients autografted after BEAC (BCNU/etoposide/cytarabine/cyclophosphamide) without consolidation chemotherapy (P = 0.015). In conclusion, post-transplant consolidation therapy is feasible and well-tolerated for patients autografted for aggressive NHL and HD and may be associated with improved progression-free survival particularly for patients with HD.

Regulation of death receptor expression and TRAIL/Apo2L-induced apoptosis by NF-kappaB.

TRAIL (tumour-necrosis factor-related apoptosis ligand or Apo2L) triggers apoptosis through engagement of the death receptors TRAIL-R1 (also known as DR4) and TRAIL-R2 (DR5). Here we show that the c-Rel subunit of the transcription factor NF-kappaB induces expression of TRAIL-R1 and TRAIL-R2; conversely, a transdominant mutant of the inhibitory protein IkappaBalpha or a transactivation-deficient mutant of c-Rel reduces expression of either death receptor. Whereas NF-kappaB promotes death receptor expression, cytokine-mediated activation of the RelA subunit of NF-kappaB also increases expression of the apoptosis inhibitor, Bcl-xL, and protects cells from TRAIL. Inhibition of NF-kappaB by blocking activation of the IkappaB kinase complex reduces Bcl-x L expression and sensitizes tumour cells to TRAIL-induced apoptosis. The ability to induce death receptors or Bcl-xL may explain the dual roles of NF-kappaB as a mediator or inhibitor of cell death during immune and stress responses.

Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1alpha.

The switch to an angiogenic phenotype is a fundamental determinant of neoplastic growth and tumor progression. We demonstrate that homozygous deletion of the p53 tumor suppressor gene via homologous recombination in a human cancer cell line promotes the neovascularization and growth of tumor xenografts in nude mice. We find that p53 promotes Mdm2-mediated ubiquitination and proteasomal degradation of the HIF-1alpha subunit of hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that regulates cellular energy metabolism and angiogenesis in response to oxygen deprivation. Loss of p53 in tumor cells enhances HIF-1alpha levels and augments HIF-1-dependent transcriptional activation of the vascular endothelial growth factor (VEGF) gene in response to hypoxia. Forced expression of HIF-1alpha in p53-expressing tumor cells increases hypoxia-induced VEGF expression and augments neovascularization and growth of tumor xenografts. These results indicate that amplification of normal HIF-1-dependent responses to hypoxia via loss of p53 function contributes to the angiogenic switch during tumorigenesis.

Increased levels of serum hepatocyte growth factor in patients with end-stage renal disease.

Blood levels of hepatocyte growth factor (HGF) have been found to be elevated in patients with chronic renal failure. The cause of the increase in this mitogen is unclear. We determined serum HGF levels in 34 patients on maintenance dialysis and ten healthy volunteers. Predialysis serum HGF levels were elevated in patients with end-stage renal disease as compared to control subjects (1.65 +/- 0.2 ng/mL vs 0.46 +/- 0.04 ng/mL, p<0.01). In addition, serum HGF levels were significantly higher in African-American dialysis patients compared to Caucasian patients (2.18 +/- 0.36ng/mL vs 1.18 +/- 0.12ng/mL, p<0.01). The observed differences could not be accounted for by variations in serum creatinine, serumalbumin, or blood pressure between the African-American and Caucasian patients. Serum HGF levels were elevated in patients with end-stage renal disease, and were higher in African-American than Caucasian patients, but the pathophysiology and significance of this finding remain unclear.

Salvage therapy for refractory chronic graft-versus-host disease with mycophenolate mofetil and tacrolimus.

Chronic graft-versus-host disease (GVHD) is a common late complication of allogeneic bone marrow transplantation (BMT) and is the principal cause of morbidity and non-relapse mortality. The improved management of acute GVHD has not translated into lower rates of chronic GVHD as older patients undergo allogeneic BMT, more patients receive unrelated or related mismatched allogeneic BMT, and donor lymphocyte infusion is increasingly used for treatment of post-BMT relapses. Patients with high risk chronic GVHD or those who fail on standard therapy have a bad prognosis. Salvage therapies have produced disappointing results. Here, we present a retrospective analysis of 26 patients where a steroid sparing synergistic combination of mycophenolate mofetil (MMF) and tacrolimus was used in refractory chronic GVHD. 46% patients showed an objective response, 11.5% had stable disease, 34.6% had progression and 7.7% were not evaluable. The combination was well tolerated. This promising preliminary result has prompted a trial to assess the safety and efficacy of this regimen in patients with chronic GVHD who have failed prior therapy and to determine if it would improve survival as well as quality of life in such patients.

Interferon-alpha-associated focal segmental glomerulosclerosis with massive proteinuria in patients with chronic myeloid leukemia following high dose chemotherapy.

BACKGROUND: Acute renal failure, with or without massive proteinuria, is a rare idiosyncratic toxicity of interferon (IFN)-alpha therapy. The authors sought to review their experience with this toxicity as well as the world literature on the subject. METHODS: The authors describe two patients with chronic myeloid leukemia treated with IFN-alpha following high dose chemotherapy who developed renal failure and proteinuria after 3 and 4 weeks of IFN-alpha therapy, respectively. Fifteen previously reported cases of renal failure and proteinuria associated with IFN-alpha therapy are also reviewed. RESULTS: Renal biopsies performed on the authors' two patients revealed focal segmental glomerulosclerosis. However, the other reported patients with IFN-alpha-associated renal failure and massive proteinuria had an assortment of pathologic findings. CONCLUSIONS: The specific renal pathology associated with proteinuria may be a consequence of the condition and not its cause; differences in renal pathology may be caused by other predisposing factors. Patients treated with IFN-alpha following high dose chemotherapy, with or without autologous transplantation, should be followed for the development of proteinuria and renal failure.

p53-mediated repression of nuclear factor-kappaB RelA via the transcriptional integrator p300.

The p53 tumor suppressor gene plays an instrumental role in transcriptional regulation of target genes involved in cellular stress responses. p53-dependent transactivation and transrepression require its interaction with p300/CBP, a coactivator that also interacts with the RelA subunit of nuclear factor-kappaB. We find that p53 inhibits RelA-dependent transactivation without altering RelA expression or inducible kappaB-DNA binding. p53-mediated repression of RelA is relieved by p300 overexpression and the increased RelA activity conferred by p53-deficiency is counteracted by either transactivation domain-deficient p300 fragments that bind RelA or a transdominant mutant of IkappaB alpha. Our results suggest that p53 can regulate diverse kappaB-dependent cellular responses.

Biological significance and molecular mechanisms of p53-induced apoptosis.

The recognition that the p53 tumour suppressor gene is frequently inactivated in human cancers has galvanized an intense pursuit of the fundamental mechanisms by which the encoded protein halts malignant transformation and tumour progression. It is now evident that p53 is a multifunctional transcription factor that is intimately involved in the cellular response to stressful stimuli such as DNA damage and hypoxia. In addition to its role in the surveillance mechanisms that arrest cell cycle progression, p53 can also trigger apoptosis in response to DNA damage or oncogenic aberrations that induce aberrant cell cycle progression. Since p53 is a critical component for DNA damage-induced apoptosis, the frequent occurrence of p53 mutations in human neoplasia provides a genetic basis for their poor response to genotoxic anticancer agents. Two recent studies offer key insights into the molecular mechanisms employed by p53 to induce cell death. One model indicates that p53 induces redox-related genes that generate reactive oxygen species and promote the oxidative degradation of mitochondrial components. The other demonstrates p53-mediated induction of DR5, a death receptor of the tumour necrosis factor receptor family, that induces death by caspase-mediated proteolysis. These insights provide an exhilarating array of possible therapeutic interventions against p53-deficient human cancers that may pay enormous dividends in the not-too-distant future.

Human herpes virus-6 encephalitis after bone marrow transplantation: successful treatment with ganciclovir.

Here we report a case that presented with sudden onset of neurological symptoms and was treated with ganciclovir. Polymerase chain reaction analysis for human herpes virus-6 (HHV-6) from his cerebrospinal fluid was later found to be positive. He subsequently recovered with minimal residual neurological symptoms. Encephalitis secondary to this virus may be more common than previously thought in patients undergoing bone marrow transplantation. This condition should be considered in the differential diagnosis of sudden onset neurological symptoms after bone marrow transplantation.

Role of thrombospondin in mesangial cell growth: possible existence of an autocrine feedback growth circuit.

Thrombospondin (TSP) is an extracellular matrix glycoprotein involved in mesangial cell (MC) adhesive and migratory function. We have studied the role of TSP in activation and proliferation of rat MC in serum-free media. TSP, in a concentration dependent manner (5 to 20 micrograms/ml), caused an increase in thymidine uptake, first detectable at 28 hours and more prominent at 48 hours. This effect was inhibited by heparin and heparan sulfate. TSP induced epidermal growth factor (EGF) secretion and significantly augmented constitutive platelet-derived growth factor-AB (PDGF-AB) secretion by MC in a concentration dependent fashion. It did not, however, induce TGF-beta, IL-1, IL-6, IL-8, or TNF-alpha production. TSP had an additive effect with exogenous EGF and PDGF on thymidine uptake. Anti-PDGF neutralizing antibody eliminated the effect of TSP on MC growth. MC displayed a single class of heparin-inhibitable TSP binding sites (Bmax 3.8 +/- 1.8 x 10(6)/cell, Kd = 80 +/- 29 nM). Based on these observations, we propose the existence of an autocrine positive feedback loop of MC proliferation involving TSP and growth factors, and regulated by heparan sulfate.

Regulation of phosphoprotein p18 in leukemic cells. Cell cycle regulated phosphorylation by p34cdc2 kinase.

p18 is a phosphoprotein that is expressed at very high levels in leukemic cells, at moderately high levels in proliferating normal lymphocytes, and at low levels in quiescent lymphocytes. Induction of terminal differentiation of leukemic cells in culture results in a decrease in cellular proliferation. These phenotypic changes are associated with rapid phosphorylation of p18, followed by a more gradual decrease in the level of its mRNA expression. More than 12 different phosphorylation products of p18 have been identified in different cells by high resolution two-dimensional polyacrylamide gel electrophoresis. Previous studies have suggested that p18 may be a substrate for protein kinase C in some cellular processes and protein kinase A in others. In this report, we show that the phosphorylation of p18 increases as cells progress toward the G2-M phases of the cell cycle in proliferating leukemic cells. We have examined the hypothesis that the putative role of p18 in cellular proliferation may be mediated by its involvement in the p34cdc2 kinase signal transduction pathway. We have produced recombinant p18 in bacterial cells and shown that it can be phosphorylated in vitro by purified p34cdc2 kinase with a stoichiometry of 0.86 mol of PO4/mol of substrate. We have used site-directed mutagenesis to demonstrate that the site of p34cdc2 phosphorylation is the serine at position 38. This same site has previously been shown to be phosphorylated in vivo in bovine brain along with another serine at position 25. The observation that p18 gets phosphorylated in the G2-M phases of the cell cycle and the demonstration that p18 is phosphorylated efficiently by p34cdc2 kinase in vitro at a residue that is also phosphorylated in vivo support the hypothesis that p18 may be a physiologic substrate for p34cdc2 kinase in vivo. We have also examined the effect of inhibiting the expression of p18 on cell cycle progression. These experiments demonstrated that antisense inhibition of the expression of p18 in K562 erythroleukemia cells is associated with a decrease in cellular proliferation and accumulation of cells in the G2-M phases of the cycle. The implications of these findings to the proposed role of p18 in the regulation of cellular proliferation are discussed.

Changes in glucose transport and transporter isoforms during the activation of human peripheral blood lymphocytes by phytohemagglutinin.

We have explored the mechanism of stimulation of glucose transport during PHA stimulation of human peripheral blood lymphocytes (HPBT) enriched in T cells. Equilibrium exchange flux of 3-O-methyl glucose (3-O-MG) was stimulated two- and fourfold at 24 and 48 h after PHA stimulation, respectively. The increase was transient in that the flux rate returned to control (unstimulated) levels by 96 h. Immunoblotting and immunoprecipitation using specific Abs revealed that resting HPBT expresses glucose transporter isoforms GLUT-2 and GLUT-3 but not GLUT-1. After PHA stimulation, GLUT-1 expression was induced predominantly in the plasma membrane, whereas GLUT-3 expression was simultaneously down-regulated. GLUT-1 expression was detectable at 24 h, peaked at 48 h, and disappeared at 96 h. The total number of glucose transporters per cell measured as the total capacity of D-glucose displaceable cytochalasin B binding did not change significantly at any time after PHA stimulation. PHA stimulation also caused expression of high affinity IL-2R and secretion of IL-2. The IL-2 secretion was transient, which peaked at 24 h, slightly preceding the GLUT-1 expression peak and disappeared at 72 h. In PHA-activated HPBT cells synchronized at G0-G1, GLUT-1 was not expressed but was rapidly induced by exposure to IL-2. This induction did not occur in the presence of cyclosporin A, which inhibits IL-2 secretion. Based on these observations, we conclude that PHA stimulation increases glucose transport partly by inducing the expression of GLUT-1 instead of GLUT-3 and that GLUT-1 expression is induced by signals generated by IL-2 binding to its high affinity receptors.

Calcium uptake during mitogenic stimulation of human lymphocytes: characterization of intracellular calcium compartments and demonstration of the presence of immunoreactive calrecticulin.

Phytohemagglutinin (PHA) stimulation of human peripheral blood lymphocytes (HPBL) rapidly increases 45Ca2+ uptake into intracellular pools. Detectable increase in 45Ca2+ uptake occurred only on exposure to mitogenic lectins but not with non-mitogenic lectins. However, intracellular free Ca2+ concentration [(Ca2+)i] increased comparably on exposure to either mitogenic or non-mitogenic lectins. Permeabilization of 45Ca2+ loaded cells revealed distinct pools of Ca2+ uptake. The highly digitonin sensitive pool #I (permeabilized by 0.02% digitonin) exchanged slowly and included a part that represented endoplasmic reticulum. Pool II was defined by lower digitonin sensitivity, had a much faster initial uptake. Pool III was digitonin-resistant and predominantly non-vesicular. During the first 120 min of PHA stimulation, significant increase in 45Ca2+ uptake occurred only into pool II. Progressive increase in uptake into pool I then occurred so that by 24 hours, this pool constituted the major fraction of PHA induced increment in total 45Ca2+ uptake. Using specific antibody to the calcium binding protein calreticulin, an analogous immunoreactive protein was detectable in resting HPBL. PHA stimulation led to a striking increase in abundance of immunoreactive calreticulin so that 24 hrs after PHA stimulation, there was a 28 and 3.4 fold increase in the amount of immunoreactive calreticulin present in the non-particulate fraction and the total particulate membrane fraction, respectively. A major part (72%) of the total cellular immunoreactive calreticulin in PHA stimulated cells at 24 hrs was released into the medium after permeabilization of lymphocytes with 0.02% digitonin, corresponding to the location of calcium uptake pool I.

Spontaneous delta- to beta-globin switching in K562 human leukemia cells.

Previous analysis of the hemoglobin phenotype of the K562 human erythroleukemia cell line showed regulated expression of the epsilon-, zeta-, gamma-, alpha-, and delta-globin genes. Expression of the beta-globin genes has not been previously detected in this cell line. In this report, we describe the isolation of a variant of the K562 cell line that actively expresses beta-globin messenger RNA (mRNA) and polypeptide and shows greatly reduced expression of the delta-globin genes. This phenotype developed spontaneously in culture while two other K562 isolates grown under the same culture conditions have not undergone the same delta- to beta-globin switch. Analysis of this unique K562 variant shows that a construct containing a beta-globin promoter is quite active upon transient transfection into these cells. This finding suggests that the activation of the endogenous beta-globin genes results from changes in the trans-acting environment of these cells. The regulation of the beta-globin genes in this variant is characterized by a paradoxical decrease in the level of beta-globin mRNA after exposure to hemin. Other globin genes of this variant are appropriately regulated and show increased expression after hemin induction. Further study of this variant may shed light on mechanisms of gene regulation that are involved in hemoglobin switching.

Direct demonstration of engraftment of human peripheral blood leukocytes in SCID mice.

One of the major problems using man-mouse chimeras is still the difficulty to demonstrate unequivocally engraftment of human cells in murine tissues. Using supravital labelling of human leukocytes with the Hoechst dye H33342, it was possible to demonstrate directly their engraftment and to assess their distribution in the tissues of the severe combined immunodeficiency (SCID) mice. The human cells can be traced for a period of 4-5 weeks. In contrast to earlier reports, combined marker and labelled-cell studies suggest that T-cell surface marker CD3 with reported specificity for human lymphocytes are indeed found, also in man-mouse chimeras, only on human cells. The ratio of B and T cells of human origin changes significantly after transfer into SCID mice and differs among various SCID tissues. The simple staining procedure using the supravital nuclear dye H33342 opens new possibilities for the study of cellular interactions and host responses of the human immunoreactive cells in an increasingly well-characterized animal model.