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.

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.

Furosemide inhibits glucose transport in isolated rat adipocytes via direct inactivation of carrier proteins.

Furosemide inhibits 3-O-methyl-D-glucose equilibrium flux in isolated adipocytes. The inhibition is saturable with an increasing concentration of furosemide and shows a noncompetitive type of kinetics. Both basal and insulin-stimulated fluxes are equally affected by the inhibition. Hydrochlorothiazide and piretanide also inhibit the flux with a similar potency, whereas bumetanide, a more potent diuretic, is much less potent. To understand the molecular basis of this inhibition, effects of furosemide on the glucose-sensitive cytochaslasin B binding activities of adipocytes were studied. Furosemide inhibits the glucose-sensitive cytochalasin B binding of both microsomal and plasma membrane preparations. For both preparations, the inhibition is time dependent and only slowly reversible, is saturable with an increasing concentration of furosemide, shows a noncompetitive type of kinetics with apparent Ki (the inhibitor concentration that gives the half-maximum effect) of 3.5 and 0.7 mM after 2 and 18 h incubation, respectively, and is essentially identical between the basal and insulin-stimulated adipocytes. The inhibition develops with a first-order rate constant of approximately 0.12/h at 4 degrees C. These results indicate that furosemide inhibits glucose transport in adipocytes by directly inactivating transport carriers of both plasma membranes and microsomal reserve pool. This inactivation of glucose carrier may play a part in the diuretic-induced glucose intolerance frequently observed during diuretic therapy.

Mechanism of mitogen-induced stimulation of glucose transport in human peripheral blood mononuclear cells. Evidence of an intracellular reserve pool of glucose carriers and their recruitment.

The present study examines the effects of phytohemagglutinin stimulation of a population of human (h) PBMC enriched in lymphocytes (hPBMC) on D-glucose displaceable cytochalasin B binding sites or medium-affinity sites (M-sites) in relation to glucose transport. Previously we have shown that M-sites are glucose transporters in hPBMC (Mookerjee, B.K., et al. 1981. J. Biol. Chem. 256:1290-1300). Equilibrium exchange of 3-O-methyl D-glucose in unstimulated cells revealed two populations with fast and slow flux rates. Phytohemagglutinin stimulates flux rates by converting part of the slow flux population to the fast flux population. M-sites occur in two distinct pools, one in plasma membrane and the other in microsomal fraction. Phytohemagglutinin treatment increases the plasma membrane pool size of M-sites with a concomitant reduction in the microsomal pool size without affecting the binding affinities or the total number of M-sites/cell. Data presented in this paper demonstrate that there are two pools of glucose transporters in these cells and phytohemagglutinin stimulation induces an energy-dependent net translocation of glucose transporters from an intracellular reserve pool to the plasma membrane, which accounts for greater than 60% of the increment in glucose transport.

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.

Human recombinant interleukin-2 is mitogenic to human lymphocytes.

Reported herein are the results of studies demonstrating that purified recombinant human interleukin-2 (hrlL-2) is a potent mitogen for lymphocytes of healthy human donors. The specificity of the hrlL-2-induced response was defined in experiments in which mitogenicity of this T cell growth-promoting lymphokine was completely abrogated by blocking the T cell membrane receptor for IL-2 with the anti-Tac monoclonal antibody. Depletion of adherent mononuclear leukocytes markedly reduced lymphocyte reactivity to hrlL-2, but the response could be fully recovered by the addition of interleukin-1 (IL-1). Increased proliferative responses were observed using a combination of hrlL-2 and a monoclonal antibody OKT3 that defines a T cell membrane antigen. These studies demonstrate that hrlL-2, as with antigens and phytomitogens, may serve as the first signal of T cell proliferation.

Chemiluminescence and lymphocyte proliferation: parallelism in collaboration between subpopulations of thymus cells for both types of responses.

Rat thymocytes respond to exposure to phytomitogens by oxidant generation as detected by chemiluminescence in presence of luminol. Maximal chemiluminescent response to a wide variety of plant lectins was obtained only after the cells had been incubated for 18 hours at 37 degrees C but not at 4 degrees C. This temperature dependence and the necessity for intact protein-and RNA-synthetic machinery during the incubation period indicate the occurrence of differentiation of thymocytes as they develop the capacity for chemiluminescent response. Furthermore, adherent-phagocytic cells play an essential collaborative role during this differentiation. A remarkable parallelism was shown to exist in the capacity of a cell subset to respond to concanavalin A by DNA synthesis and the ability of the same subset to respond by chemiluminescence. The latest-sedimenting small lymphocytes after velocity sedimentation of thymus cells develop the capacity for DNA-synthetic as well as chemiluminescent responses to concanavalin A only if allowed to collaborate with a population of early-sedimenting adherent-phagocytic cells.

Tuberous sclerosis with striking renal involvement in a family.

We describe five cases of tuberous sclerosis in members of one family, all having renal involvement but with differences in age of onset and mode of presentation. Clinical, laboratory, and pathologic features helpful in diagnosing this condition and in distinguishing it from polycystic kidney disease and renal neoplasms are stressed. Tuberous sclerosis should always be considered in differential diagnosis of patients with multiple cystic renal lesions, particularly when age of onset of symptoms ranges from infancy to adult life in different members of one family. Absence both of specific glomerular or tubular lesions in ultrastructure and of major abnormalities in renal tubular function supports the existing concept that replacement of nephrons by hamartomatous lesions is the cause of progressive renal failure.

Role of endogenous prostaglandins in volume expansion and during furosemide infusion in conscious dogs.

The renal effects of two structurally dissimilar inhibitors of prostaglandin synthesis (Meclofenamate and RO-20-5720) were studied in conscious, chronically instrumented dogs during mild volume expansion and during a constant infusion of furosemide. When either inhibitor was administered following volume expansion, urinary excretion of PGE2 and urine flow rate were reduced by more than 50%. In contrast, renal plasma flow fell by less than 10% while glomerular filtration rate, sodium excretion, and plasma renin activity (PRA) were unchanged. In separate studies, infusion of furosemide increased renal plasma flow, urine flow rate, sodium excretion, PRA, and urinary excretion of PGE2, while glomerular filtration rate decreased. Administration of inhibitors of prostaglandin synthesis during constant infusion of furosemide reduced the urinary excretion of PGE2 to control levels, as renal plasma flow and glomerular filtration rate fell below control level. Despite these hemodynamic alterations, the furosemide-induced diuresis and increase in PRA were only partly attenuated by prostaglandin inhibition. It is concluded that in conscious dogs, intrarenal prostaglandins modulate urine flow rate during mild volume expansion and play a major role in mediating the renal hemodynamic effects of furosemide.

Renal prostaglandins and the regulation of blood pressure and sodium and water homeostasis.

In addition to its well known prohypertensive role in various states of experimental and human hypertension, the kidney has also been shown to exert an antihypertensive "endocrine" function. According to this hypothesis, certain forms of experimental and human hypertension might not solely be the result of an excess in the activity of such renal pressor systems as the renin-angiotensin system and the sympathetic nervous system, but might also result from an absolute or relative deficiency of intra-renal vasodilator antihypertensive factors which might allow pressor systems to act unopposed to produce peripheral arteriolar vasoconstriction and sustained hypertension. At least four factors have been characterized in the kidney of various animal species and man which might be responsible for such an antihypertensive function. These are (1) the renomedullary prostaglandins (PGs), (2) the renomedullary antihypertensive neutral lipid, (3) antirenin phospholipid and (4) the renal kinins. This review is restricted to an examination of the possibility that the vasodepressor renomedullary prostaglandins (PGA and/or PGE) may, at least in part, mediate the so-called antihypertensive function of the kidney and participate in the regulation of renal blood flow and natriuresis by physiologic antagonism of various renal vasoconstrictor stimuli such as the renal renin-angiotensin and the sympathetic nervous systems.

Functional characteristics of monocytes. 1. Essential role in the transformational response of human blood lymphocytes to phytomitogens.

Phagocytic glass-adherent mononuclear cells in peripheral human blood (morphologically monocytes) have been shown to play an essential role in lymphocyte transformational response to phytomitogens. Lymphocytes from peripheral blood have been exhaustively depleted of monocytes by successive treatments, first with opsonized iron particles with eventual magnetic elimination of phagocytic cells, followed by filtration of the same population through packed glass-sand columns. This subpopulation, enriched in T lymphocytes and containing around 4% B lymphocytes, does not transform in response to concanavalin A, phytohemagglutinin, or pokeweek mitogen unless viable monocytes are added to the culture. Supernatant fluids from monocyte-enriched human blood leukocyte cultures can substitute for monocytes in restoring response. Interaction of the phytomitogen with lymphocyte, while essential, is not sufficient to trigger transformation, requiring a second signal provided by the monocyte probably via release of soluble mediators.

Influence of separation techniques on the distribution and function of lymphocyte subpopulations. A comparison of three techniques.

The effect, if any, of three different lymphocyte separation techniques on the composition and functional characteristics of the purified cell suspensions has been studied. Each separation technique was shown to yield a cell population with highly specific and reproducible characteristics. The Ficoll-Hypaque technique led to good lymphocyte yields but low yields of sheep erythrocyte-rosetting (E-rosetting) T cells, and the separated cell population responded least well to phytohemagglutinin. The glass sand filtration technique led to lowest overall yield of small lymphocytes and of EAC-rosetting cells. There was significantly lower total yield of E-rosetting T cell as well, but the separated lymphocyte suspension had excellent purity, had relatively high percentage of E-rosetting T cells, and they responded extremely well to phytohemagglutinin (PHA) and pokeweed mitogen (PWM). The Technicon separation involving magneticremoval of phagocytic cells by exposure to iron particles consistently led to large yields of small lymphocytes with good purity, the largest total harvests of E-rosetting T cells, as well as EAC-rosetting cells while the separated population had the highest percentage of E-rosetting cells and responded very well to PHA and PWM. These results show that lymphocyte losses during purification are not nonspecific and that the choice of the separation technique profoundly affects the characteristics of the purified lymphocyte population obtainable.

Functional characteristics of monocytes. II. Further observations on lymphocyte-monocyte collaboration in the response to phytomitogens.

Monocytes play a crucial role in the response of lymphocytes to phytomitogens. This role is evident during the very early events after exposure to the mitogen. Thus, significant lymphocyte transformation does occur when monocytes are removed from the culture within a few hours after exposure to mitogen, and the presence of monocytes after the initial period is no longer necessary. Monocytes appear to be required for the early increase in protein synthesis and burst in RNA synthesis following exposure to the mitogen. Monocytes may help in lymphocyte responses partly by longer range, by way of release of soluble humoral factors into culture supernatant fluids. Temperature dependence of the effect of pulse exposure of lymphocytes to these supernatants raises the possibility that membrane receptors on these cells to humoral factors released by monocytes may exist. Moncytes do not appear to act primarily by preserving the viability of lymphocytes in culture.

Effects of diphtheria toxin and other exotoxins on oxidant generation by human and murine phagocytes.

Bacterial exotoxins such as diphtheria toxin (D.T.), staphylococcal alpha toxin and Leucocidin can powerfully activate granulocytes and macrophages as detected by production of chemiluminescence in presence of Luminol. Production of superoxide by granulocytes and of prostaglandin E2 in macrophages is also stimulated by D.T. In contrast with the known resistance of rodent parenchymal cells to the diphtheria toxin, human and rodent leucocytes have similar sensitivities to D.T.

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.

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.

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.

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.

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.