Early Versus Delayed Autologous Stem Cell Transplantation and Interferon Maintenance in Multiple Myeloma: Single-Center Experience of 18 Years.

BACKGROUND: Autologous stem cell transplantation (ASCT) has become the mainstay of 1st-line treatment in younger patients with multiple myeloma (MM), but statistical confirmation of its superiority over other therapies, especially in the era of novel agents, is still lacking. METHODS: We reviewed the results of all 548 myeloma ASCTs performed in our institute over the past 18 years. RESULTS: More than one-half of the patients had access to novel agents before their transplantations. Although the age of the transplanted patients increased significantly over the years, treatment-related mortality (TRM) was remarkably low, especially in 1st-line transplanted patients (100-day TRM, 0.3%). The median overall survival (OS) of the entire cohort was 98.4 months. Patients transplanted within 12 months from the start of their therapy had significantly better responses than those having delayed ASCT (complete response rate, 58.1% vs 46.8%; P = .016) and significant post-ASCT progression-free survival (PFS) benefit (30.2 [26.1-34.3] mo vs 23.3 [16.8-29.8] mo; P = .036), but we found no significant overall survival difference. The results were similar in patients treated with or without novel agents before ASCT. During a period of time, interferon maintenance was our standard approach to post-ASCT maintenance. Our analysis showed not only a significant PFS advantage with interferon, but also a highly significant overall survival benefit (150.4 [105.1-195.8] mo vs 86.1 [72.5-99.7] mo; P = .003). CONCLUSIONS: Our findings demonstrate that delayed ASCT can be feasible in selected patients.

The potential role of HLA-DRB1*11 in the development and outcome of haematopoietic stem cell transplantation-associated thrombotic microangiopathy.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a serious complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT) with high mortality rate. We retrospectively studied the frequency, clinical and genetic associations and prognostic effect of TA-TMA, in a total of 425 consecutive adult patients, who underwent allo-HSCT for a malignant haematological condition between 2007 and 2013 at our single centre. TA-TMA developed in 19% of the patients. Unrelated donor type (P<0.001), acute GvHD grades II-IV (P<0.001), myeloablative conditioning regimens (P=0.003), tacrolimus-based GvHD prophylaxis (P=0.003), CMV infection (P=0.003) and carriership for HLA-DRB1*11 (P=0.034) were associated with the development of TA-TMA. Survival was adversely affected by the presence of TA-TMA (P<0.001). Among patients with TA-TMA, the outcome of HLA-DRB1*11 carriers was significantly better compared with non-carriers (P=0.003). As a new finding, our observations suggest that the presence of HLA-DRB1*11 antigen contributes to the development of TA-TMA and affects the outcome.

Selective effect of noncytotoxic blocking alloantibodies produced after platelet transfusions on MLC, and mitogen and soluble antigen-induced responses of human lymphocytes.

The transfusion of blood components (buffy coat and platelets) may induce characteristic alloimmune response or suppressive regulation that have, in certain cases, a beneficial effect on allograft survival. The blocking effect of the sera of donors immunized with platelets on mixed lymphocyte culture and on the response of lymphocytes to mitogen as well as soluble antigen (PPD, tetanus toxoid) stimulation was studied. Six sera from 7 volunteers displayed a strong and significant nonspecific MLC blocking effect that was detectable on the 10th day following the second platelet transfusion (PT). Incubation of isolated stimulator and effector cell population with this "blocking sera" showed that the latter are involved in the mediation of suppression in the MLC test. This inhibitory effect is associated with the serum IgG fraction lacking any correlation with either class I or class II specific cytotoxic antibodies. Selective blocking behavior was found on transformation activity induced by mitogens or soluble antigens. Thus, sera of platelet-transfused volunteers decreases the responsiveness of lymphocytes to phytohemagglutinin, while the response to concanavalin A, tetanus toxoid, and PPD was not suppressed. Separate treatment of T and B lymphocyte populations and monocytes with the blocking sera showed that only T and B lymphocytes are targets, and not the monocytes for the inhibition in the case of PHA-induced proliferation. Indirect evidence may support the notion that MLC-inhibiting and FcR-blocking antibodies may be analog products of a regulatory alloimmune response induced by leukocytes that are partially responsible for the beneficial transfusion effect in organ transplantation.

Functional and immunogenetic characterization of FcR-blocking antibody.

The characteristics and functional importance of FcR-blocking antibodies and their production were investigated after immunization with whole blood, "buffy coat" and purified platelets. We studied the presence of FcR-blocking antibody in haemodialyzed, transfused patients waiting for kidney transplantation, and we found strong correlation between the blocking effect and better graft survival. We suggest that this blocking antibody does not attack FcR as primary target. Investigation of blocking activity of ten different immune sera on 50 healthy panel cells showed that target antigen has some polymorphic varieties. On basis of family studies it seems that the target antigen is not linked to HLA haplotype. The blocking effect of sera could be removed by absorption of CD8+ cells, B lymphocytes, platelets and granulocytes, but not with erythrocytes, monocytes, CD8- cells and NK cells.

Non-cytotoxic blocking antibodies and suppressor cells induced by donor-specific transfusions in healthy volunteers and potential kidney transplant recipients.

Donor-specific transfusion (DST)-induced immunosuppression plays a significant role in clinical and experimental transplantation. To clarify the mechanism of suppression on alloreactivity the suppressor cell induction and the non-cytotoxic blocking antibody production and importance was studied in 15 healthy volunteers and 3 kidney transplant recipients (KTR) after DST on mixed lymphocyte culture (MLC). Significant decrease of anti-donor MLC response was found in all of KTR and in 12 cases of the 15 transfused volunteers. Of 18 cases, 15 had blocking factors in their post-DST serum which strongly (52-91%) inhibited the MLC response. The IgG fractions isolated from the "blocking sera" were responsible for this inhibition. Eighty-one percent of non-cytotoxic blocking antibodies affected the responder cells in MLC and reacted with third party responder cells as well. Both buffy coat and platelet transfusions evoked production of the non-specific blocking antibodies. Our data strongly suggest that DST induces not only the differentiation of suppressor cells and production of anti-idiotypic antibodies, but also the appearance of non-specific, non-cytotoxic antibodies, which may participate in the cell-mediated immune suppression of the alloimmune reactivity.

Differences in non-MHC restricted cytotoxic activities of human peripheral blood lymphocytes after transfusion with allogeneic leukocytes or platelets possessing class I and/or class II MHC molecules.

MHC-unrestricted cytotoxic activity of peripheral blood lymphocytes (PBL) from 4-6 healthy donors was investigated before and after transfusion with allogeneic leukocytes or platelets. Natural killer and lectin-dependent cellular cytotoxicity (LDCC) of PBL was tested against K562 and Raji target cells in a 4-h and 16-h 51Cr-release assay, respectively. After allotransfusion with leukocytes, we found increased cytotoxic activity of each donor's PBL against all the three targets on day 3 or 7. The highest non-specific cytotoxic activity was detected against the relatively NK resistant Raji target cells. The increase of cytotoxic activity was lowest against the LDCC target (PHA-treated Raji) cells. On the contrary, no changes in cytotoxic activity against any targets were observed after allotransfusion with platelets (possessing class I HLA antigens but no HLA class II molecules). Our results suggest that HLA class II molecules, presumably by inducing immune responses, are essential for activation/generation of non-specific killing of tumor targets after leukocyte transfusion. Thrombocytes, known to be less immunogenic than leukocytes, are not effective in in vivo enhancing of non-specific cytotoxicity. Cellular activation of PBL following leukocyte allotransfusion was confirmed by detection of elevated serum neopterin and beta-2-microglobulin levels on day 3. This was not the case after platelet allotransfusion. In addition, the expression of ICAM-1 antigen (as a molecule involved directly in MHC-unrestricted cytotoxicity) was also found to be increased in two donors' PBL on day 3 after leukocyte transfusion in contrast to transfusion with platelets.

Allogeneic bone marrow transplantation for acute leukemia in adults.

From September 1993 to August 1997, 24 consecutive adult acute leukemia patients (20 AML and 4 ALL) received allogeneic bone marrow transplant (21 sibling, 1 twin, 2 MUD). The probability of 3 year leukemia free survival is 19/24 (79%), the transplant related mortality is 2/24 (8%), the relapse rate is 3/24 (13%). The median follow up period is 34 months (range 7-51). Three AML patients with high probability of TRM received a special radiation-free conditioning regimen (mitobronitol/cytarabine/ cyclophosphamide) originally described by Kelemen et al in CML patients for decreasing transplant related complications. All the three patients are alive and disease free over 3 years.

Autologous stem cell transplantation for malignant lymphomas.

From December 1995 to April 1998, 20 consecutive adult patients suffering from chemosensitive relapse of Hodgkin's or non-Hodgkin lymphoma (11 Hodgkin, 9 non-Hodgkin Lymphoma) received autologous stem cell transplantation. The median follow up period is 15 months (range 6-28). The overall survival is 18/20 (90 %), the event free survival is 13/20 (65%). None of the patients died of transplant related cause.

Bone marrow transplantation in non-malignant disorders.

From January 1992 to December 1997, 21 consecutive patients (14 SAA, 3 SCID, 1 Fanconi anemia, 1 Diamond-Blackfan anemia, 1 mucolipidosis and 1 mucopolysaccharidosis type I.) were transplanted (16 HLA-id. family, 2 MUD and 3 haploidentical family donors) in a single center. The median follow up period is 41 months (range 7-76). The probability of 3.5 year overall disease free survival is 14/21 (67%), the transplant related mortality is 4/21 (19%). All the SCID patients are alive and disease free. 3 SAA patients had signs of fungal infection prior to transplant. They died in spite of intensive antifungal treatment resulting reduced DFS for SAA to 71%. Two patients with lysosomal storage disorders (mucolipidosis and MPS I.) rejected the haploidentical T-cell depleted graft 1 and 11 months after transplant, respectively. In 2 cases non-engraftment occured, both patients were retransplanted successfully.

Time course of quantitative morphological changes of the autophagic-lysosomal compartment of murine seminal vesicle epithelial cells under the influence of vinblastine.

Changes of the autophagic-lysosomal compartment (ALC) of the murine seminal vesicle epithelial cells were monitored by electron microscopic morphometry during 36 h following a single 10 mg/kg bw dose of vinblastine sulfate (VBL), a widely used tool to cause an accumulation of the autophagic vacuoles (AVs). Three morphologically distinct subcompartments of the ALC, i.e. early autophagic vacuoles (AV1) being presumably prelysosomal autophagosomes, advanced AVs (AV2) containing material under degradation and dense bodies (DB) were defined. The ALC and its subcompartments expanded after VBL in a two-phase reaction. The first subcompartments to react significantly were AV1 and AV2 (at 30 min) followed by DBs with a 30 min delay. The ALC then ceased to grow until the 90th min when a second expansion phase started peaking around 8 h with a cytoplasmic volume fraction 15 times larger than in the untreated control. This second growth was entirely brought about by the expansion of the two AV subcompartments. After 8 h the volume fraction of both AV1 and AV2 decreased to cause a gradual regression of the ALC. AV1, however, already ceased to expand as early as after 6 h, i.e. during the last 2 h of the expansion phase of the ALC. Comparison of this time curve with the one we previously measured in mouse liver shows considerable differences between the two cell types. The growth curves of the AV subcompartments in our experiment along with others' kinetic data obtained in steady state cells not treated with VBL show that segregation (= formation of AV1) is possibly stimulated by VBL in our system.

[Intrauterine management of Rh-alloimmunization]. / Az Rh-alloimmunizáció intrauterin kezelése.

The authors report the strategy of invasive management of Rh alloimmunisation in pregnancy. From the 34 pregnancies 6 were monitored by amniocenteses, 11 by fetal blood sampling, and 4 with combination of the two above mentioned diagnostic procedures. In 13 cases the fetuses were treated with intrauterine intravascular blood transfusions. All the procedures were ultrasound guided. The fetal blood sampling and the transfusions were carried out by puncturing the umbilical vein or artery. For transfusions, maternal blood was used in case of identical blood type, otherwise adult Rh negative, filtered, washed, irradiated blood was transfused. They report the complications as well, giving the cause of their fetal losses in details. There were no maternal complications observed. Out of the 34 pregnant women 25 had healthy newborns, which number is acceptable in this disease with a very high mortality rate. The authors underline that the technique of fetal blood sampling and intrauterine transfusion if needed is necessary in the management of Rh alloimmunised pregnancies.

[A new radiation-free conditioning in bone marrow transplantation and dibromo-mannitol therapy in chronic myeloid leukemia]. / Uj, sugárzásmentes csontvelö-transzplantációs kondicionáló kezelés dibróm-mannitollal krónikus myeloid leukaemiában.

A new, radiation-free, conditioning protocol, containing the original Hungarian mitobronitol (DBM) (DBM/ cytosine arabinoside/cyclosphosphamide) has been applied to 36 chronic myeloid leukemia (CML) patients followed by bone marrow transplantation (BMT) from HLA identical sibling donors between 1990-1997. In spite of some prognostically disadvantageous factors (half of them were above 40 years, 10 out of 36 patients were in accelerated phase, the disease history was longer than 2 years in average) the overall survival (30/36) and the leukemia free survival rate (26/36) were in accordance with the best international results. Transplantation-related toxicity was remarkably reduced in comparison to bone marrow transplantation performed by total body irradiation/cyclophosphamide (TBI/Cy) or busulphan/cyclophosphamide (Bu/Cy) conditioning protocols. Acute graft versus host disease was present in lower percentage (9/36) and the number of serious cases was only 2/36. Chronic GVH disease, generally known to be associated with antileukemic effect (GVL), occurred in 25 of cases. Early haematological relapse among the 34 patients with functioning graft occurred in 6 patients which rate is slightly higher than reported after TBI/Cy or Bu/Cy conditioning treatment. There was no relapse among patients transplanted within one year post-diagnosis and patients having CML with accelerated phase. The leukemia free post-transplant period was in association with the chronic GVH disease and full chimeric state.

Complement activation in thrombotic thrombocytopenic purpura.

BACKGROUND: Ultra-large von Willebrand factor and deficiency of its cleaving protease are important factors in the events leading to thrombotic microangiopathy; however, the mechanisms involved are only partly understood. Whereas pathological activation of the alternative complement pathway is linked to atypical hemolytic uremic syndrome, the role of complement activation in thrombotic thrombocytopenic purpura (TTP) is unknown. The aim of this study was to investigate whether signs of complement activation are characteristic of TTP. PATIENTS AND METHODS: Twenty-three patients with TTP (18 women, median age 38 years) and 17 healthy controls (13 women, median age 38 years) were included. Complement parameters (C3, Factors H, I, B and total alternative pathway activity) together with complement activation fragments (C3a) or complexes (C1rs-INH, C3bBbP, sC5b9) were measured by ELISA or RID. ADAMTS13 activity and anti-ADAMTS13 inhibitory antibodies were measured by the VWF-FRET73 assay. RESULTS: Increased levels of C3a, and SC5b9 were observed in TTP during acute episodes, as compared with healthy controls. Decreased complement C3 levels indicative of complement consumption occurred in 15% of acute TTP patients. Significant decrease of complement activation products C3a and SC5b9 was observed during plasma exchange (PEX). The sustained presence of anti-ADAMTS13 inhibitory antibodies in complete remission was associated with increased complement activation. CONCLUSION: These data document in an observational study the presence of complement activation in TTP. Further investigation is needed to determine its potential pathogenetic significance.

Diagnosis and classification of hemolytic uremic syndrome: the Hungarian experience.

BACKGROUND: Hemolytic uremic syndrome (HUS) is a rare disease with various etiologies, making the identification of the specific forms and appropriate treatment difficult. Therefore, clinical and laboratory data from these patients need to be analyzed in national and international registries. Herein we have described 47 Hungarian HUS patients with detailed laboratory and clinical data obtained between 2008 and 2010. METHODS: Blood samples and clinical data of 47 patients with HUS diagnosed according to characteristic clinical signs were submitted for diagnostic evaluation, including complement protein and genetic analysis, measurement of ADAMTS13 activity and antibody analysis against O157LPS and factor H. RESULTS: There were 8 patients with typical diarrhea-positive HUS; 13 with atypical HUS (aHUS) and 26 with secondary HUS/thrombotic thrombocytopenic purpura group characterized by signs of complement consumption and decreased ADAMTS13 activity. Thus, decreased total alternative pathway activity is a promising diagnostic parameter with good sensitivity for aHUS. CONCLUSIONS: These observations highlight the requirement for multiple diagnostic tests together with clinical data to identify the specific cause of HUS. Because the long-term prognosis of aHUS, eg, graft survival after renal transplantation, may vary according to the molecular etiology, it is important for all affected patients to undergo a detailed molecular diagnosis of the disease. There is a clear clinical need for the development and application of novel assay in this field to allow more rapid efficient diagnosis of patients who undergo a first episode of HUS.

Immunologic consequences of blood transfusion and their clinical manifestations.

Aspects of clinical immunology, in the context of transfusion medicine, became highlighted in the last decade as a consequence of the accelerating expansion of basic immunology, immunogenetics and molecular biology. In addition sophisticated new technologies, which were capable of producing pure and safe blood products, attracted more attention to research and monitor the consequences of transfusion. These technologies also had obvious effects on supportive hematological therapy. The transfusion of blood components follows the rules of organ transplantation: when there is a mismatch between the donor and the recipient, the transfusion has the potential to induce various kinds of immune response against alloantigens. Antigen-compatible transfusions that involve major and rare blood groups are in almost all cases mismatched with respect to various polymorphic systems expressed on the cellular blood components. These include histocompatibility leukocyte antigens (HLA), tissue-specific and differentiation alloantigens, and, in the case of plasma, immunoglobulins, complement components, heat shock proteins, and shedded soluble membrane alloantigens. Clinical manifestations of alloimmune responses are typically deleterious. For example, immediate antigen-antibody binding and its consequences as secondary activations are paralleled by the nonhaemolytic febrile reaction, HLA sensitization can lead to a state of platelet refractoriness and inconvenient clinical symptoms. In certain immunogenetic situations and in immunodeficient patients graft-versus-host disease can be induced by blood products that contain live lymphocytes. Leukocyte filtration techniques are widely used to avoid most but not all of these harmful side effects of blood component therapy. In contrast to these harmful side effects in certain immunogenetic conditions, alloantigens that are expressed on various blood products can elicit an advantageous suppression of the immune response in the recipient. In the context of kidney transplantation this is termed the 'beneficial transfusion effect', and typically results in the prolongation of the graft's survival. In cases of recurrent habitual abortion and IgG therapy associated with certain autoimmune diseases, immunization with leukocytes specifically takes advantage of this phenomenon. To date the beneficial transfusion effect is not fully understood. In certain cases of malignancies or gastrointestinal surgeries this suppression of immune regulation that is induced by transfusion can worsen the clinical state either by permitting the spread of the tumor or by allowing severe infections to proceed unchecked. In conclusion it is imperative to monitor the immunological consequences of transfusion in order to deter the disadvantageous side effects. Taking advantage of the 'beneficial transfusion effect' may also provide a new means for immune therapy using the various blood products.

Blast colony forming cells in umbilical cord blood: frequency and correlation to other haemopoietic progenitors.

Umbilical cord blood has been suggested as a potential source of haemopoietic stem cells for clinical transplantation. Assay of stroma adherent blast colony forming cells (CFU-BL), as a cell type that may predict marrow repopulation, has already been suggested to predict the outcome of bone marrow transplantation. In the present experiments the frequency of CFU-BL in plastic non adherent mononuclear cell fraction (PNAMNC) of umbilical cord blood was studied. If PNAMNC from cord blood was co-incubated with a pre-established stromal layer from normal bone marrow a strict linear correlation between panned PNAMNC and blast colonies were observed. Frequency of CFU-BL in cord blood was significantly lower than that in bone marrow. In cord blood, CFU-GEMM frequency was found to be comparable to that of bone marrow, CFU-GM and BFU-E frequency was reduced. A good correlation between CFU-BL and BFU-E frequency was found both in cord blood and bone marrow. On the other hand, irrespective of their source, correlation between CFU-BL and CFU-GEMM or CFU-GM was weak.