Age-adjusted high-dose chemotherapy followed by autologous stem cell transplantation or conventional chemotherapy with R-MP as first-line treatment in elderly primary CNS lymphoma patients - the randomized phase III PRIMA-CNS trial.

BACKGROUND: Older primary central nervous system lymphoma (PCNSL) patients have an inferior prognosis compared to younger patients because available evidence on best treatment is scarce and treatment delivery is challenging due to comorbidities and reduced performance status. High-dose chemotherapy and autologous stem cell transplantation (HCT-ASCT) after high-dose methotrexate (MTX)-based immuno-chemotherapy has become an increasingly used treatment approach in eligible elderly PCNSL patients with promising feasibility and efficacy, but has not been compared with conventional chemotherapy approaches. In addition, eligibility for HCT-ASCT in elderly PCNSL is not well defined. Geriatric assessment (GA) may be helpful in selecting patients for the best individual treatment choice, but no standardized GA exists to date. A randomized controlled trial, incorporating a GA and comparing age-adapted HCT-ASCT treatment with conventional chemotherapy is needed. METHODS: This open-label, multicenter, randomized phase III trial with two parallel arms will recruit 310 patients with newly diagnosed PCNSL > 65 years of age in 40 centers in Germany and Austria. The primary objective is to demonstrate that intensified chemotherapy followed by consolidating HCT-ASCT is superior to conventional chemotherapy with rituximab, MTX, procarbazine (R-MP) followed by maintenance with procarbazine in terms of progression free survival (PFS). Secondary endpoints include overall survival (OS), event free survival (EFS), (neuro-)toxicity and quality of life (QoL). GA will be conducted at specific time points during the course of the study. All patients will be treated with a pre-phase rituximab-MTX (R-MTX) cycle followed by re-assessment of transplant eligibility. Patients judged transplant eligible will be randomized (1:1). Patients in arm A will be treated with 3 cycles of R-MP followed by maintenance therapy with procarbazine for 6 months. Patients in arm B will be treated with 2 cycles of MARTA (R-MTX/AraC) followed by busulfan- and thiotepa-based HCT-ASCT. DISCUSSION: The best treatment strategy for elderly PCNSL patients remains unknown. Treatments range from palliative to curative but more toxic therapies, and there is no standardized measure to select patients for the right treatment. This randomized controlled trial will create evidence for the best treatment strategy with the focus on developing a standardized GA to help define eligibility for an intensive treatment approach. TRIAL REGISTRATION: German clinical trials registry DRKS00024085 registered March 29, 2023.

Optimizing MATRix as remission induction in PCNSL: de-escalated induction treatment in newly diagnosed primary CNS lymphoma.

BACKGROUND: Primary diffuse large B-cell lymphoma (DLBCL) of the central nervous system (PCNSL) is a rare disorder with an increasing incidence over the past decades. High-level evidence has been reported for the MATRix regimen (high-dose methotrexate (HD-MTX), high-dose AraC (HD-AraC), thiotepa and rituximab) followed by high-dose chemotherapy and autologous stem cell transplantation (HCT-ASCT) supporting this approach to be considered a standard therapy in newly diagnosed PCNSL patients ≤ 70 years. However, early treatment-related toxicities (predominantly infectious complications), occurring in up to 28% per MATRix cycle, diminish its therapeutic success. Furthermore, sensitivity to first-line treatment is an independent prognostic factor for improved overall survival (OS) in PCNSL. Thus, patients achieving early partial remission (PR) after 2 cycles of MATRix might be over-treated with 4 cycles, in the context of consolidation HCT-ASCT. METHODS: This is an open-label, multicentre, randomized phase III trial with two parallel arms. 326 immunocompetent patients with newly diagnosed PCNSL will be recruited from 37 German, 1 Austrian and 12 UK sites. Additional IELSG (International Extranodal Lymphoma Study Group) sites are planned. The objective is to demonstrate superiority of a de-escalated and optimised remission induction treatment strategy, followed by HCT-ASCT. Randomization (1:1) will be performed after completion of all screening procedures. Patients in Arm A (control treatment) will receive 4 cycles of MATRix. Patients in Arm B (experimental treatment) will receive a pre-phase (R/HD-MTX), followed by 2 cycles of MATRix. Patients in both arms achieving PR or better will proceed to HCT-ASCT (BCNU, thiotepa). The primary endpoint of the study is event-free-survival (EFS), defined as time from randomization to premature end of treatment due to any reason, lymphoma progression or death whichever occurs first. Secondary endpoints include OS, progression free survival (PFS), toxicity, neurocognitive impairment and quality of life. Minimal follow-up is 24 months. DISCUSSION: Current treatment options for PCNSL in patients ≤ 70 years have improved remarkably over recent years. However, the potential efficacy benefits are offset by an increased incidence of short-term toxicities which can impact on treatment delivery and hence on survival outcomes. In patients ≤ 70 years with newly diagnosed PCNSL addressing the need to reduce treatment-related toxicity by de-escalating and optimising the induction phase of treatment, is a potentially attractive treatment strategy. TRIAL REGISTRATION: German clinical trials registry DRKS00022768 registered June 10th, 2021.

High-dose chemotherapy with autologous haemopoietic stem cell transplantation for newly diagnosed primary CNS lymphoma: a prospective, single-arm, phase 2 trial.

BACKGROUND: High-dose methotrexate-based chemotherapy is standard for primary CNS lymphoma, but most patients relapse. High-dose chemotherapy with autologous stem cell transplantation (HCT-ASCT) is supposed to overcome the blood-brain barrier and eliminate residual disease in the CNS. We aimed to investigate the safety and efficacy of HCT-ASCT in patients with newly diagnosed primary CNS lymphoma. METHODS: In this prospective, single-arm, phase 2 trial, we recruited patients aged 18-65 years with newly diagnosed primary CNS lymphoma and immunocompetence, with no limitation on clinical performance status, from 15 hospitals in Germany. Patients received five courses of intravenous rituximab 375 mg/m(2) (7 days before first high-dose methotrexate course and then every 10 days) and four courses of intravenous high-dose methotrexate 8000 mg/m(2) (every 10 days) and then two courses of intravenous rituximab 375 mg/m(2) (day 1), cytarabine 3 g/m(2) (days 2 and 3), and thiotepa 40 mg/m(2) (day 3). 3 weeks after the last course, patients commenced intravenous HCT-ASCT (rituximab 375 mg/m(2) [day 1], carmustine 400 mg/m(2) [day 2], thiotepa 2 × 5 mg/kg [days 3 and 4], and infusion of stem cells [day 7]), irrespective of response status after induction. We restricted radiotherapy to patients without complete response after HCT-ASCT. The primary endpoint was complete response at day 30 after HCT-ASCT in all registered eligible patients who received at least 1 day of study treatment. This trial is registered at ClinicalTrials.gov, number NCT00647049. FINDINGS: Between Jan 18, 2007, and May 23, 2011, we recruited 81 patients, of whom two (2%) were excluded, therefore we included 79 (98%) patients in the analysis. All patients started induction treatment; 73 (92%) commenced HCT-ASCT. 61 (77·2% [95% CI 66·1-86·6]) patients achieved a complete response. During induction treatment, the most common grade 3 toxicity was anaemia (37 [47%]) and the most common grade 4 toxicity was thrombocytopenia (50 [63%]). During HCT-ASCT, the most common grade 3 toxicity was fever (50 [68%] of 73) and the most common grade 4 toxicity was leucopenia (68 [93%] of 73). We recorded four (5%) treatment-related deaths (three [4%] during induction and one [1%] 4 weeks after HCT-ASCT). INTERPRETATION: HCT-ASCT with thiotepa and carmustine is an effective treatment option in young patients with newly diagnosed primary CNS lymphoma, but further comparative studies are needed. FUNDING: University Hospital Freiburg and Amgen.

High-dose chemotherapy and autologous stem cell transplant compared with conventional chemotherapy for consolidation in newly diagnosed primary CNS lymphoma--a randomized phase III trial (MATRix).

BACKGROUND: Primary central nervous system lymphoma (PCNSL) is a highly aggressive Non-Hodgkin lymphoma (NHL) with rising incidence over the past 30 years in immunocompetent patients. Although outcomes have improved, PCNSL is still associated with inferior prognosis compared to systemic NHL. Many questions regarding the optimal therapeutic approach remain unanswered. METHODS/DESIGN: This is a randomized, open-label, international phase III trial with two parallel arms. We will recruit 250 patients with newly diagnosed PCNSL from approximately 35 centers within the networks of the German Cooperative PCNSL study group and the International Extranodal Lymphoma Study Group. All enrolled patients will undergo induction chemotherapy consisting of 4 cycles of rituximab 375 mg/m(2)/d (days 0 & 5), methotrexate 3.5 g/m(2) (d1), cytarabine 2 × 2 g/m(2)/d (d2-3), and thiotepa 30 mg/m(2) (d4) every 21 days. All patients will undergo stem-cell harvest after the second cycle. After 4 cycles of induction chemotherapy, patients achieving partial or complete response will be centrally randomized to 2 different consolidation treatments: (A) conventional-dose immuno chemotherapy with rituximab 375 mg/m(2) (d0), dexamethasone 40 mg/d (d1-3), etoposide 100 mg/m(2)/d (d1-3), ifosfamide 1500 mg/m(2)/d (d1-3) and carboplatin 300 mg/m(2) (d1) (R-DeVIC) or (B) high-dose chemotherapy with BCNU (or busulfan) and thiotepa followed by autologous stem cell transplantation (HCT-ASCT). The objective is to demonstrate superiority of HCT-ASCT compared to R-DeVIC with respect to progression-free survival (PFS, primary endpoint). Secondary endpoints include overall survival (OS), treatment response and treatment-related morbidities. Minimal follow-up after treatment completion is 24 months. DISCUSSION: The rationale for consolidation treatment in PCNSL is to eliminate residual lymphoma cells and to decrease the risk for relapse. This can be achieved by agents crossing the blood brain barrier either applied at conventional doses or at high doses requiring autologous stem cell support. HCT-ASCT has been shown to be feasible and highly effective in patients with newly-diagnosed PCNSL. However, it is unclear whether HCT-ASCT is really superior compared to conventional-dose chemotherapy after an intensified antimetabolites-based immunochemotherapy in patients with newly-diagnosed PCNSL. To answer this question, we designed this investigator initiated randomized phase III trial. TRIAL REGISTRATION: German clinical trials registry DRKS00005503 registered 22 April 2014 and ClinicalTrials.gov NCT02531841 registered 24 August 2015.

Rab8 binding to immune cell-specific adaptor LAX facilitates formation of trans-Golgi network-proximal CTLA-4 vesicles for surface expression.

Despite playing a central role in tolerance, little is known regarding the mechanism by which intracellular CTLA-4 is shuttled from the trans-Golgi network to the surfaces of T cells. In this context, Ras-related GTPase Rab8 plays an important role in the intracellular transport, while we have previously shown that CTLA-4 binds to the immune cell adaptor TRIM in T cells. In this study, we demonstrate that CTLA-4 forms a multimeric complex comprised of TRIM and related LAX that in turn binds to GTP bound Rab8 for post-Golgi transport to the cell surface. LAX bound via its N terminus to active GTP-Rab8, as well as the cytoplasmic tail of CTLA-4. TRIM required LAX for binding to Rab8 in a complex. Wild-type LAX or its N terminus (residues 1 to 77) increased CTLA-4 surface expression, whereas small interfering RNAs of Rab8 or LAX or disruption of LAX/Rab8 binding reduced numbers of CTLA-4-containing vesicles and its coreceptor surface expression. LAX also promoted the polarization of CTLA-4 and the reorientation of the microtubule-organizing center to the site of T-cell receptor engagement. Our results identify a novel CTLA-4/TRIM/LAX/Rab8 effector complex in the transport of CTLA-4 to the surfaces of T cells.

CTLA-4 activation of phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death.

The balance of T-cell proliferation, anergy and apoptosis is central to immune function. In this regard, co-receptor CTLA-4 is needed for the induction of anergy and tolerance. One central question concerns the mechanism by which CTLA-4 can induce T-cell non-responsiveness without a concurrent induction of antigen induced cell death (AICD). In this study, we show that CTLA-4 activation of the phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death. CTLA-4 ligation induced PI 3K activation as evidenced by the phosphorylation of PKB/AKT that in turn inactivated GSK-3. The level of activation was similar to that observed with CD28. CTLA-4 induced PI 3K and AKT activation also led to phosphorylation of the pro-apoptotic factor BAD as well as the up-regulation of BcL-XL. In keeping with this, CD3/CTLA-4 co-ligation prevented apoptosis under the same conditions where T-cell non-responsiveness was induced. This effect was PI 3K and PKB/AKT dependent since inhibition of these enzymes under conditions of anti-CD3/CTLA-4 co-ligation resulted in cell death. Our findings therefore define a mechanism by which CTLA-4 can induce anergy (and possibly peripheral tolerance) by preventing the induction of cell death.

CTLA-4 trafficking and surface expression.

The T-cell co-receptor cytotoxic T-cell antigen 4 (CTLA-4) has a strong inhibitory role as shown by the lymphoproliferative phenotype of CTLA-4-deficient mice. Despite its potent effects on T-cell function, CTLA-4 is primarily an intracellular antigen whose surface expression is tightly regulated by restricted trafficking to the cell surface and rapid internalisation. Recently, several signalling molecules such as Trim, PLD, ARF-1 and TIRC7 have been described to be involved in the transport of CTLA-4 to the cell surface. Minor changes in surface expression levels have major effects on the outcome of T-cell activation. Optimal regulation of CTLA-4 surface expression is crucial for the balance of stimulatory and inhibitory signals to maximize protective immune responses while maintaining immunological tolerance and preventing autoimmunity.

Adaptor SKAP-55 binds p21 activating exchange factor RasGRP1 and negatively regulates the p21-ERK pathway in T-cells.

While the adaptor SKAP-55 mediates LFA-1 adhesion on T-cells, it is not known whether the adaptor regulates other aspects of signaling. SKAP-55 could potentially act as a node to coordinate the modulation of adhesion with downstream signaling. In this regard, the GTPase p21(ras) and the extracellular signal-regulated kinase (ERK) pathway play central roles in T-cell function. In this study, we report that SKAP-55 has opposing effects on adhesion and the activation of the p21(ras) -ERK pathway in T-cells. SKAP-55 deficient primary T-cells showed a defect in LFA-1 adhesion concurrent with the hyper-activation of the ERK pathway relative to wild-type cells. RNAi knock down (KD) of SKAP-55 in T-cell lines also showed an increase in p21(ras) activation, while over-expression of SKAP-55 inhibited activation of ERK and its transcriptional target ELK. Three observations implicated the p21(ras) activating exchange factor RasGRP1 in the process. Firstly, SKAP-55 bound to RasGRP1 via its C-terminus, while secondly, the loss of binding abrogated SKAP-55 inhibition of ERK and ELK activation. Thirdly, SKAP-55-/- primary T-cells showed an increased presence of RasGRP1 in the trans-Golgi network (TGN) following TCR activation, the site where p21(ras) becomes activated. Our findings indicate that SKAP-55 has a dual role in regulating p21(ras)-ERK pathway via RasGRP1, as a possible mechanism to restrict activation during T-cell adhesion.

T cell receptor-interacting molecule acts as a chaperone to modulate surface expression of the CTLA-4 coreceptor.

The costimulatory molecule CTLA-4 is a potent downregulator of T cell responses. Although localized mostly in intracellular compartments, little is understood regarding the mechanism that regulates its transport to the cell surface. In this study, we demonstrated that the adaptor TRIM (T cell receptor-interacting molecule) bound to CTLA-4 in the trans Golgi network (TGN) and promoted transport of CTLA-4 to the surface of T cells. Increased TRIM expression augmented surface CTLA-4 expression, and pulse-chase analysis showed a more rapid transport of CTLA-4 to the cell surface. A reduction of TRIM expression by small hairpin RNAs reduced the expression of surface CTLA-4. This resulted in a more localized pattern of CTLA-4 in the TGN. Altered CTLA-4 expression by TRIM was accompanied by corresponding changes in coreceptor-mediated effects on cytokine production and proliferation. Our findings identify a role for TRIM as a chaperone in regulating CTLA-4 expression and function by enhancing CTLA-4 transport to the surface of T cells.

CTLA-4 up-regulation of lymphocyte function-associated antigen 1 adhesion and clustering as an alternate basis for coreceptor function.

Although cytotoxic T lymphocyte antigen-4 (CTLA-4) negatively regulates T cell activation, the full range of functions mediated by this coreceptor has yet to be established. In this study, we report the surprising finding that CTLA-4 engagement by soluble antibody or CD80 potently up-regulates lymphocyte function-associated antigen 1 (LFA-1) adhesion to intercellular adhesion molecule-1 (ICAM-1) and receptor clustering concurrent with IL-2 inhibition. This effect was also observed with CTLA-4 ligation and not with other coreceptors. T cell antigen receptor (TcR)-induced lymphocyte function-associated antigen 1 function was also dependent on CTLA-4 expression as observed with reduced adhesion/clustering on CTLA-4(-/-) primary T cells. CTLA-4 up-regulated adhesion was mediated by regulator for cell adhesion and polarization type 1 (Rap-1) as shown by anti-CTLA-4-induced Rap-1 activation as well as Rap-1-N17 blockade and Rap-1-V12 mimicry of adhesion/clustering. Our findings identify a potent role for CTLA-4 in directing integrin adhesion and provide an alternate mechanism to account for aspects of CTLA-4 function in T cell immunity.

Coupling of contact sensitizers to thiol groups is a key event for the activation of monocytes and monocyte-derived dendritic cells.

Strong contact sensitizers are able to induce distinct signal transduction mechanisms in antigen-presenting cells by coupling to cell proteins. The predominant target structures of haptens are thought to be thiol and amino groups in cysteine and lysine residues. We studied whether coupling of small reactive chemicals to thiol or amino groups might be responsible for the activation of monocytes and mature monocyte-derived dendritic cells. Human peripheral blood mononuclear cells were stimulated in vitro with subtoxic concentrations of the strong haptens 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone and 2, 4, 6-trinitrochlorobenzene, the thiol-reactive reagents N-hydroxymaleimide and N-ethylmaleimide, as well as the amino-reactive compounds sulfosuccinimidyl acetate and 2-iminothiolane. Flow cytometric quantification of tyrosine phosphorylation in CD14+ monocytes showed that 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone, 2, 4, 6-trinitrochlorobenzene, N-hydroxymaleimide, and N-ethylmaleimide but not sulfosuccinimidyl acetate and 2-iminothiolane strongly induced this process. Tyrosine phosphorylation induced by 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone and 2, 4, 6-trinitrochlorobenzene was completely prevented in the presence of cysteine but not lysine, suggesting a competitive mechanism between cysteine and sulfhydryl groups of cell proteins. Using the mouse ear swelling test N-hydroxymaleimide could be classified as a significant contact allergen in comparison to 2, 4, 6-trinitrochlorobenzene, whereas no sensitizing potential became apparent for sulfosuccinimidyl acetate and 2-iminothiolane. Western blot analysis on monocytes and mature monocyte-derived dendritic cells confirmed the flow cytometric data for tyrosine phosphorylation and demonstrated a selective capacity of haptens and thiol-reactive compounds to activate ERK1/2 mitogen-activated protein kinase. Our data show that strong affinity of a small reactive chemical toward thiol groups is important for the activation of monocytes and monocyte-derived dendritic cells and can support the process of sensitization.

Thiol antioxidants block the activation of antigen-presenting cells by contact sensitizers.

Strong contact sensitizers are able to induce signal transduction mechanisms such as tyrosine phosphorylation and activation of MAP kinases in antigen-presenting cells. We studied the capacity of different antioxidants (ascorbic acid, alpha-tocopherol, pyrrolidine dithiocarbamate, N-acetylcysteine, and glutathione) to block the increase in tyrosine phosphorylation in human monocytes seen after stimulation with strong contact sensitizers. Human peripheral blood mononuclear cells were stimulated with 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone in the presence or absence of these antioxidants. The total amount of membrane-associated phosphotyrosine in CD14+ cells was quantified using flow cytometric techniques. Complete inhibition of tyrosine phosphorylation was noticed when cells were stimulated in the presence of N-acetylcysteine or glutathione. Using N-acetylcysteine as inhibitor similar results were obtained for cells stimulated with formaldehyde, thimerosal methyldibromoglutaronitrile, diphenylcyclopropenone, p-phenylenediamine, toluene-2,5-diamine, and 2,4-dinitrofluorobenzene. By use of a trinitrophenyl-specific monoclonal antibody it was shown that N-acetylcysteine as well as cysteine prevents the binding of 2,4,6-trinitrochlorobenzene to proteins in monocytes and monocyte-derived mature dendritic cells. Furthermore, the capacity of N-acetylcysteine to block the activation of p38 and ERK1/2 MAP kinases by 2,4,6-trinitrochlorobenzene was demonstrated. The radical scavengers ascorbic acid and alpha-tocopherol as well as the nuclear factor kappaB inhibitor pyrrolidine dithiocarbamate failed to prevent the increase in tyrosine phosphorylation. Our data present evidence that reactive oxygen species as well as transcription factor nuclear factor kappaB seem to be unimportant for the induction of tyrosine phosphorylation by contact sensitizers. On the other hand, protection of thiol groups using compounds with free sulfhydryl groups is very effective to block this process. This finding may have implications for prevention of occupational sensitization to strong contact allergens.

Activation and translocation of p38 mitogen-activated protein kinase after stimulation of monocytes with contact sensitizers.

Recently we described the induction of tyrosine phosphorylation by contact sensitizers as an early molecular event during the activation of antigen- presenting cells. In this study, the role of the p38 mitogen-activated protein kinase for the activation of human monocytes after exposure to four structurally unrelated contact sensitizers was analyzed in comparison with the irritant benzalkonium chloride and an inductor of oxidative stress (H2O2) using immunofluorescence, Western blotting, and enzyme-linked immunosorbent assay techniques. Bio chemical analysis revealed a translocation of p38 from the cytoplasm to the detergent-resistant cell fraction only upon stimulation with contact sensitizers. The activity of p38 was studied by quantification of its phosphorylated active form with a specific antibody and by kinase assay. Although all stimulants used in this study led to the activation of p38, a translocation to the detergent-resistant fraction as well phosphorylation of the mitogen-activated protein kinase dependent transcription factor Elk-1 was induced only by contact sensitizers. Evidence for a functional relevance of mitogen-activated protein kinase activation was provided by measurement of the hapten-induced production of the proinflammatory cytokine interleukin-1beta. Its release was inhibited by blocking p38-mediated signaling using the imidazole compounds SB203580 and SB202190. These data show that contact sensitizers are strong activators of the p38 mitogen-activated protein kinase. Although activation of this stress-associated pathway has been reported for many other stimuli, a unique translocation of p38 from the cytoplasm to the detergent-resistant fraction seems to be a specific event during hapten-induced activation of antigen-presenting cells.

JAK/STAT pathways are not involved in the direct activation of antigen-presenting cells by contact sensitizers.

JAK/STAT pathways are described as the major mechanisms by which cytokine receptors transduce intracellular signals. The signalling mechanisms in antigen-presenting cells (APC) in the sensitization phase of contact hypersensitivity are poorly understood. The aim of this study was to clarify whether well-established JAK/STAT signalling pathways might be activated directly by contact sensitizers as described previously for tyrosine kinases and some MAP kinases. As a model of epidermal APC, human monocytes and human monocyte-derived dendritic cells were stimulated with the structurally unrelated contact sensitizers MCI/MI, thimerosal, TNCB and formaldehyde. The phosphorylation states of the transcription factors STAT1, STAT3, STAT4, STAT5 and STAT6 were determined by Western blot analysis using phosphospecific antibodies. In contrast to the positive controls performed with the cytokines IFN-gamma, IL-10, IFN-alpha, GM-CSF and IL-4, no significant increase in the phosphorylation of STAT molecules was recognized in hapten-treated cells. These results suggest that contact allergens do not directly activate common JAK/STAT pathways. Therefore the activation of APC in the early sensitization phase of contact hypersensitivity by haptens does not involve signals normally delivered by JAK-associated cytokine receptors.