Determinants of lenalidomide response with or without erythropoiesis-stimulating agents in myelodysplastic syndromes: the HOVON89 trial.

A randomized phase-II study was performed in low/int-1 risk MDS (IPSS) to study efficacy and safety of lenalidomide without (arm A) or with (arm B) ESA/G-CSF. In arm B, patients without erythroid response (HI-E) after 4 cycles received ESA; G-CSF was added if no HI-E was obtained by cycle 9. HI-E served as primary endpoint. Flow cytometry and next-generation sequencing were performed to identify predictors of response. The final evaluation comprised 184 patients; 84% non-del(5q), 16% isolated del(5q); median follow-up: 70.7 months. In arm A and B, 39 and 41% of patients achieved HI-E; median time-to-HI-E: 3.2 months for both arms, median duration of-HI-E: 9.8 months. HI-E was significantly lower in non-del(5q) vs. del(5q): 32% vs. 80%. The same accounted for transfusion independency-at-week 24 (16% vs. 67%), but similar in both arms. Apart from presence of del(5q), high percentages of bone marrow lymphocytes and progenitor B-cells, a low number of mutations, absence of ring sideroblasts, and SF3B1 mutations predicted HI-E. In conclusion, lenalidomide induced HI-E in patients with non-del(5q) and del(5q) MDS without additional effect of ESA/G-CSF. The identified predictors of response may guide application of lenalidomide in lower-risk MDS in the era of precision medicine. (EudraCT 2008-002195-10).

Post-transfusion purpura in a woman with acute myeloid leukemia.

Post-transfusion purpura (PTP) is a rare, but severe transfusion reaction in which both donor and autologous platelets are sequestered due to immunization against HPA-1a antigens in HPA-1a negative recipients (HPA: human platelet antigens). We describe a patient who developed PTP during induction therapy for acute myeloid leukaemia. The pitfalls, delays in diagnosing and therapy options of this serious transfusion reaction are discussed.

Sequence analysis of EBV DNA isolated from mouth washings and PBMCs of healthy individuals and blood of EBV-LPD patients.

BACKGROUND: Lymphoproliferative disorder (LPD) caused by Epstein-Barr virus (EBV) is a severe complication of bone marrow transplantation. The EBV strain causing LPD is of either donor or recipient origin, however, available data are limited to only a small number of cases. To obtain solid evidence, comparison of the EBV strain that caused the EBV-LPD with pre-stem cell transplantation (SCT) EBV strains of donor and recipient is imperative. Available techniques rely on the production of EBV transformed lymphoblastoid cell lines and lack sensitivity. OBJECTIVE: The aim of this study was to develop a simple method for EBV sequence analysis on mouth washings (MWs) and peripheral blood mononuclear cells (PBMCs). STUDY DESIGN: EBV DNA was extracted from MWs and PBMCs that were collected from 20 healthy individuals. DNA was used for sequence analysis, using a polymerase chain reaction for the C-terminus of the LMP-1 gene. RESULTS: In seropositive individuals EBV DNA could be detected in 11/14 (79%) MWs and in 13/14 (93%) PBMC samples. Sequence analysis showed that in 11 out of 14 (79%) healthy individuals sequence patterns could be established. In these 11 healthy individuals 13 sequence patterns could be detected. Eleven of these 13 patterns (84.6%) were unique. These results encouraged us to explore the feasibility of this method on EBV DNA isolated from plasma from 9 EBV-LPD patients at time of EBV reactivation. In 7 EBV-LPD patients 8 sequence patterns were detected. Six out of 8 sequence patterns (75%) were unique. CONCLUSION: Our method is suitable for strain identification and we intend to use this technique to evaluate EBV origin in EBV-LPD patients.

Epstein-Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell transplantation (SCT) and quantitatively predicts EBV-lymphoproliferative disease following T-cell--depleted SCT.

Reactivation of the Epstein-Barr virus (EBV) after allogeneic stem cell transplantation (allo-SCT) may evoke a protective cellular immune response or may be complicated by the development of EBV-lymphoproliferative disease (EBV-LPD). So far, very little is known about the incidence, recurrence, and sequelae of EBV reactivation following allo-SCT. EBV reactivation was retrospectively monitored in 85 EBV-seropositive recipients of a T-cell--depleted (TCD) allo-SCT and 65 EBV-seropositive recipients of an unmanipulated allo-SCT. Viral reactivation (more than 50 EBV genome equivalents [gEq]/mL) was monitored frequently by quantitative real-time plasma polymerase chain reaction until day 180 after SCT. Probabilities of developing viral reactivation were high after both unmanipulated and TCD-allogeneic SCT (31% +/- 6% versus 65% +/- 7%, respectively). A high CD34(+) cell number of the graft appeared as a novel significant predictor (P =.001) for EBV reactivation. Recurrent reactivation was observed more frequently in recipients of a TCD graft, and EBV-LPD occurred only after TCD-SCT. High-risk status, TCD, and use of antithymocyte globulin were predictive for developing EBV-LPD. Plasma EBV DNA quantitatively predicted EBV-LPD. The positive and negative predictive values of a viral load of 1000 gEq/mL were, respectively, 39% and 100% after TCD. Treatment-related mortality did not differ significantly between TCD and non-TCD transplants, but the incidence of chronic graft-versus-host disease was significantly less in TCD patients. It is concluded that EBV reactivation occurs frequently after TCD and unmanipulated allo-SCT, especially in recipients of grafts with high CD34(+) cell counts. EBV-LPD, however, occurred only after TCD, and EBV load quantitatively predicted EBV-LPD in recipients of a TCD graft. (Blood. 2001;98:972-978)

Tetramer-based quantification of cytomegalovirus (CMV)-specific CD8+ T lymphocytes in T-cell-depleted stem cell grafts and after transplantation may identify patients at risk for progressive CMV infection.

Recovery of cytomegalovirus (CMV)-specific T-cell-mediated immunity after allogeneic hematopoietic stem cell transplantation (SCT) is critical for protection against CMV disease. The study used fluorochrome-conjugated tetrameric complexes of HLA-A2 molecules loaded with the immunodominant NLVPMVATV (NLV) peptide derived from the CMV protein pp65 to quantify A2-NLV-specific CD8+ T cells in partially T-cell-depleted grafts administered to 27 HLA-A*0201+ patients and to monitor recovery of these T cells during the first 12 months after SCT. None of the 9 CMV-seronegative patients became infected with CMV, whereas 14 of 18 CMV-seropositive patients developed CMV antigenemia after SCT. CMV-seropositive recipients of grafts from CMV-seronegative donors required more preemptive treatment with ganciclovir (GCV) than those of grafts from CMV-seropositive donors (3 [1-6] versus 1 [0-3] courses, respectively; P =.009). The number of A2-NLV-specific CD8+ T cells in the grafts correlated inversely with the number of preemptive GCV courses administered (r = -0.61; P =.01). None of the 9 CMV-seronegative patients mounted a CMV-specific immune response as measured by monitoring A2-NLV-specific CD8+ T cells after SCT. Thirteen of 14 CMV-seropositive patients without CMV disease recovered these T cells. In spite of preemptive GCV treatment, CMV disease developed in 4 patients, who all failed to recover A2-NLV-specific CD8+ T cells after SCT (P =.002). Thus, enumeration of HLA-restricted, CMV-specific CD8+ T cells in the grafts and monitoring of these T cells after SCT may constitute a rapid and sensitive tool to identify SCT recipients at risk for developing CMV disease.

Molecular quantification of viral load in plasma allows for fast and accurate prediction of response to therapy of Epstein-Barr virus-associated lymphoproliferative disease after allogeneic stem cell transplantation.

Epstein-Barr virus lymphoproliferative disease (EBV-LPD) following allogeneic stem cell transplantation (allo-SCT) has a poor prognosis. We used a sensitive real-time polymerase chain reaction (PCR) assay for quantitative detection of EBV-DNA in plasma and serially measured EBV-DNA levels to assess the response to treatment in allo-SCT recipients with EBV-LPD. Fourteen allo-SCT recipients with EBV-LPD who received a T cell-depleted (TCD) sibling (n = 5) or matched unrelated donor (n = 9) graft were monitored from the time of EBV-LPD diagnosis, during therapy and assessment of clinical response. Seven patients had complete responses of EBV-LPD to therapy, of whom 21% (3 out of 14) survived beyond 6 months from EBV-LPD diagnosis. Clinically responding patients showed a rapid decline of EBV-DNA plasma levels within 72 h from the start of therapy. In contrast, all clinical non-responders showed an increase of EBV-DNA levels. Absolute EBV-DNA levels at the time of EBV-LPD diagnosis did not predict for response, but the pattern of EBV-DNA levels within 72 h from the start of therapy (> 50% decrease versus increase) strongly predicted for clinical response (P = 0.001). Quantitative monitoring of EBV-DNA levels from the start of and during therapy for EBV-LPD rapidly and accurately predicts for response to therapy as early as within 72 h. It may thus provide a powerful tool to adjust and select treatment in individuals with EBV-LPD following allo-SCT.

Increased transplant-related morbidity and mortality in CMV-seropositive patients despite highly effective prevention of CMV disease after allogeneic T-cell-depleted stem cell transplantation.

We evaluated the efficacy, toxicity, and outcome of preemptive ganciclovir (GCV) therapy in 80 cytomegalovirus (CMV)-seropositive patients allografted between 1991 and 1996 and compared their outcome to 35 seronegative patients allografted during the same period. Both cohorts were comparable with respect to diagnosis and distribution of high- versus standard-risk patients. All patients received a stem cell graft from an HLA-identical sibling donor, and grafts were partially depleted of T cells in 109 patients. Patients were monitored for CMV antigenemia by leukocyte expression of the CMV-pp65 antigen. Fifty-two periods of CMV reactivation occurring in 30 patients were treated preemptively with GCV. A favorable response was observed in 48 of 50 periods, and only 2 patients developed CMV disease: 1 with esophagitis and 1 with pneumonia. Ten of 30 treated patients developed GCV-related neutropenia (less than 0.5 x 10(9)/L), which was associated with a high bilirubin at the start of GCV therapy. Overall survival at 5 years was 64% in the CMV-seronegative cohort and 40% in the CMV-seropositive cohort (P =.01). Increased treatment-related mortality accounted for inferior survival. CMV seropositivity proved an independent risk factor for developing acute graft-versus-host disease, and acute graft-versus-host disease predicted for higher treatment-related mortality and worse overall survival in a time-dependent analysis. We conclude that, although CMV disease can effectively be prevented by preemptive GCV therapy, CMV seropositivity remains a strong adverse risk factor for survival following partial T-cell-depleted allogeneic stem cell transplantation.

Expression of T cell activation antigen CD134 (OX40) has no predictive value for the occurrence or response to therapy of acute graft-versus-host disease in partial T cell-depleted bone marrow transplantation.

CD134 (OX40) is a member of the tumor necrosis factor family which is expressed by activated T lymphocytes. CD134 expression on T cells was monitored during the first 35 days post-transplant in 14 patients, receiving either an HLA-identical sibling bone marrow transplant (BMT), a matched unrelated transplant (MUD-BMT) or an autologous peripheral blood progenitor cell transplant (PBPCT). The sibling and unrelated grafts were partially depleted of T cells. CD134 expression on CD4+ T cells peaked between 7 and 14 days after BMT, with a mean peak value of 45% of CD4+ cells (range 26-70%) over all three patient groups. The observed pattern of CD4+ CD134+ expression, an increase during the first 2 weeks post-BMT followed by a gradual decline towards values of 15-40%, was similar in all groups. No difference in the kinetics of CD134 expression by CD4+ T cells was observed between the patients that did or did not develop graft-versus-host disease (GVHD), nor did the clinical effect of any treatment given for GVHD correlate with alterations in CD134 expression by CD4+ T cells. Absolute CD4+,CD134+ T cell numbers showed a more rapid increment after autologous PBPCT than after sibling or MUD transplants. We conclude that expression of CD134+ by CD4+ T lymphocytes cannot serve as a surrogate marker for allo-reactivity. CD134+ expression may reflect lymphocyte regeneration, rather than alloreactivity.

An uncommon cause of anaemia in ulcerative colitis.

A 56-year-old woman is reported with severe ulcerative colitis who developed a corticosteroid-resistant haemolytic anaemia. The pathogenesis and treatment options in ulcerative-colitis-related haemolytic anaemia are discussed.

The effects of recombinant human erythropoietin on hemostasis and fibrinolysis in hemodialysis patients.

Thromboembolism might complicate the treatment of patients with chronic renal failure with Recombinant Human Erythropoietin (ReHuEPO). In order to detect prothrombotic changes, a number of hemostatic and fibrinolytic parameters was determined during ReHuEPO treatment of fifteen chronic hemodialysis patients (mean age 47.1 years; ten females, five males). To avoid the influence of hemoconcentration and/or dilution, the patients were kept normovolemic, using the method of echography of the inferior vena cava diameter. In a first group of eight patients, we investigated platelet count and function. During ReHuEPO, a significant rise of hematocrit (19 +/- 3 to 34 +/- 5%, p < 0.001) was observed. Bleeding time shortened (7'33'' +/- 3'39'' to 3'41'' +/- 3'19''; p < 0.001) and platelet count increased (222 +/- 45 to 254 +/- 49 10 9/l; p < 0.005). The initial negative in vitro spontaneous platelet reactivity became positive in two of them, whereas the decrease in ADP threshold in the whole group (2.0 +/- 0.1 to 1.10 +/- 0.4 mumol, p < 0.02) indicated an increased induced platelet reactivity. In all patients prothrombotic changes were observed. The protein-C antigen and protein-C activity and the total and free protein-S antigen decreased significantly. The plasminogen activator inhibitor (PAI) activity in the whole group did not change significantly (6.4 +/- 4.1 to 5.4 +/- 4.8 AU/ml). However, in the patients with fistula thrombosis (n = 3), higher values in all test points were found compared to those without thrombosis.

Recombinant human erythropoietin and its effects on macro- and microcirculation during normovolemia. A physiological study of hemodynamics, fluid status and skin microcirculation.

In 9 chronic hemodialysis patients, treated with recombinant human erythropoietin (rHuEpo), longitudinal studies were performed to investigate possible changes in macro- and microcirculatory parameters during normovolemia, as assessed echographically by determining the inferior vena cava diameter and adjusting dialysis dry weight. Hematocrit increased from 19 +/- 4 to 33 +/- 5% (p less than 0.001). Systemic vascular resistance increased from 1,020 +/- 259 to 1,283 +/- 245 dyn/s/cm-5 (p less than 0.02), while mean arterial pressure remained unchanged. Cardiac index decreased (4.9 +/- 1.4 to 3.8 +/- 0.9 liters/min/m2; p less than 0.02), caused by a decrease in heart rate (87 +/- 21 to 75 +/- 16 beats/min; p less than 0.02) and stroke index (59.9 +/- 15.2 to 51.0 +/- 10.7 ml/m2; p less than 0.02). Red blood cell volume increased (468 +/- 105 to 858 +/- 203 ml/m2; p less than 0.001) and plasma volume decreased inversely ([125I]-albumin; 2,008 +/- 338 to 1,664 +/- 225 ml/m2; p less than 0.001), whereas total blood volume remained unaltered (2,476 +/- 397 to 2,518 +/- 352 ml/m2; n.s.). Total body weight increased (57.8 +/- 12 to 62.1 +/- 12 kg; p less than 0.02), indicative of an anabolic effect of rHuEpo therapy. Skin capillary circulation as measured by transcutaneous oxymetry at 37 degrees C skin temperature impaired, reflected by the increase of the time to peak after arterial occlusion (82 +/- 21 to 121 +/- 25 s; p less than 0.02). The reactive hyperemic response following the release of occlusion showed a significant increase at high hematocrit (10.7 +/- 4.2 to 16.6 +/- 5.3 mm Hg; p less than 0.02), whereas resting transcutaneous Po2 values showed a slight but not significant increase (2.3 +/- 1.3 to 4.7 +/- 3.3 mm Hg; n.s.). The high number of pathological capillaries in hemodialyzed patients might be an additional factor for the increase in systemic vascular resistance.