The impact of the age of HLA-identical siblings on mobilization and collection of PBSCs for allogeneic hematopoietic cell transplantation.

With the increasing age of patients undergoing allogeneic hematopoietic cell transplantation (HCT), the age of matched related sibling donors (MRDs) is expected to increase. Donor safety and the impact of donors' age on mobilization, collection of peripheral hematopoietic progenitor cells (HPCs), subsequent engraftment and the incidence of GVHD were retrospectively analyzed. A total of 167 patients received HCT from an MRD. Median donors' age was 48 years (67 (40%) donors were ≥50 years including 34 donors ≥60 years). Side effects under mobilization and apheresis were age independent. Grafts from donors <50 years contained more CD34+ cells (median 9 × 10(6)/kg recipient's body weight (RBW)) compared with older donors (median 5.9 × 10(6)/kg RBW) (P<0.0005), whereas harvests from donors ≥60 years contained more natural killer (NK) cells (P=0.003). Engraftment occurred at a median of 12 days after HCT irrespective of donors' age. Increasing age of MRD did not preclude successful mobilization, collection of HPC and engraftment. In the context of more NK cells in grafts from elderly donors, the impact of donors' age on outcome after HCT warrants further studies. Although short-term toxicities of apheresis were not increased with increasing age, long-term donor safety remains an important issue.

Monitoring of WT1 expression in PB and CD34(+) donor chimerism of BM predicts early relapse in AML and MDS patients after hematopoietic cell transplantation with reduced-intensity conditioning.

Relapse of malignant disease remains the major complication in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) after hematopoietic cell transplantation (HCT) with reduced-intensity conditioning (RIC). In this study, we investigated the predictive value of disease-specific markers (DSMs), donor chimerism (DC) analysis of unsorted (UDC) or CD34(+) sorted cells and Wilms' tumor gene 1 (WT1) expression. Eighty-eight patients with AML or MDS were monitored after allogenic HCT following 2 Gy total-body irradiation with (n=84) or without (n=4) fludarabine 3 × 30 mg/m(2), followed by cyclosporin A and mycophenolate mofetil. DSMs were determined by fluorescence in situ hybridization (FISH) and WT1 expression by real-time polymerase chain reaction. Chimerism analysis was performed on unsorted or CD34(+) sorted cells, by FISH or short tandem repeat polymerase chain reaction. Twenty-one (24%) patients relapsed within 4 months after HCT. UDC, CD34(+) DC and WT1 expression were each significant predictors of relapse with sensitivities ranging from 53 to 79% and specificities of 82-91%. Relapse within 28 days was excluded almost entirely on the basis of WT1 expression combined with CD34(+) DC kinetics. Monitoring of WT1 expression and CD34(+) DC predict relapse of AML and MDS after RIC-HCT.

BCR-ABL transcripts are early predictors for hematological relapse in chronic myeloid leukemia after hematopoietic cell transplantation with reduced intensity conditioning.

Kinetics of BCR-ABL transcript elimination and its prognostic implications on relapse were analyzed in patients with chronic myeloid leukemia (CML) after reduced intensity hematopoietic cell transplantation (HCT). In all, 19 CML patients were conditioned with 2 Gy total-body irradiation in combination with (n=14) or without (n=3) fludarabine 3 x 30 mg/m(2) (Flu) or 4.5 Gy total lymphoid irradiation (TLI) with Flu and OKT3 3 x 5 mg (n=2) and were treated with cyclosporine (CSP) and mycophenolate mofetil after allogeneic HCT. BCR-ABL transcripts were analyzed by nested RT-PCR and Taqman((R)) RT-PCR on days +28, +56 and +84 after HCT and were evaluated for their association with relapse. Of the 19 patients, 14 achieved sustained remissions of which six had a negative RT-PCR 28 days after HCT. Five patients relapsed +41, +54, +57, +136 and +234 days after HCT. Predictors for relapse were advanced disease stage (P=0.02) and slow reduction of BCR-ABL transcripts at day 28 (P=0.006) and day 56 (P=0.047) post-transplant. We conclude that a complete clearance of BCR-ABL transcripts is achievable within 4 weeks from HCT even after minimal conditioning and that early kinetics of BCR-ABL transcripts significantly correlate with the probability of hematological relapse.

Hematopoietic cell transplantation from related and unrelated donors after minimal conditioning as a curative treatment modality for severe paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disorder caused by a somatic mutation of the X-linked phosphatidylinositol glycan class A gene. Allogeneic hematopoietic cell transplantation (HCT) after high-dose conditioning is the only curative treatment; however, it is associated with high treatment-related mortality. Here, we report on allogeneic HCT for PNH after minimal conditioning. Seven adult patients with high-risk PNH underwent peripheral blood HCT from HLA-A-, -B-, -C-, -DRB1-, and -DQB1-matched related (n = 2) and unrelated (n = 5) donors. Conditioning included fludarabine 30 mg/m(2)/d on days -4 to -2 and 2 Gy of total body irradiation on day 0. After HCT, patients were given immunosuppressive therapy with oral cyclosporine starting on day -3 and mycophenolate mofetil starting on day 0. All 7 patients attained durable engraftment. After 28 days, a median of 77% (range, 53%-96%) T-cell donor chimerism was found in bone marrow and peripheral blood. T-cell chimerism increased to 91% (range, 76%-100%) on day +180 and to 100% in all surviving patients after 12 months. All 7 patients attained complete remissions of their disease. Four patients are alive 13 to 38 months after HCT. Three patients died of treatment-related mortality, 1 because of complications after acute pancreatitis and multiorgan failure, 1 because of infection related to chronic graft-versus-host disease (GVHD), and 1 because of bleeding after liver biopsy for late subacute/chronic GVHD. Allogeneic HCT from related and unrelated donors after minimal conditioning is a new and potentially curative option for patients with advanced PNH.

FISH for BCR-ABL on interphases of peripheral blood neutrophils but not of unselected white cells correlates with bone marrow cytogenetics in CML patients treated with imatinib.

Interphase fluorescence in situ hybridization (I-FISH) for the BCR-ABL translocation performed on peripheral blood (PB) white cells has been suggested as a surrogate for conventional bone marrow (BM) cytogenetics for monitoring patients with chronic myeloid leukemia (CML). I-FISH is faster, less costly, and does not require BM aspiration. For patients treated with interferon-alpha (IFN), a good correlation between the two methods has been demonstrated in several though not all studies. However, imatinib mesylate (STI571) has largely replaced IFN as the standard drug treatment for CML, raising the question if the results obtained in IFN-treated patients are applicable to patients on imatinib. We therefore compared the two methods in patients on imatinib and patients on other therapies, mainly IFN (collectively referred to as nonimatinib therapies). Our results demonstrate that the correlation between I-FISH and cytogenetics is much weaker in patients on imatinib than in patients on nonimatinib therapies. Correction of the I-FISH values for the proportion of lymphocytes barely improved the correlation, probably as a result of unpredictable proportions of Philadelphia-positive B cells. By contrast, I-FISH of PB neutrophils was much better correlated with BM cytogenetics. We conclude that I-FISH on unselected PB white cells is not suitable for monitoring patients on imatinib.

High Bad and Bax mRNA expression correlate with negative outcome in acute myeloid leukemia (AML).

The search for molecular markers in AML that allow prediction of outcome has recently focused on genes involved in the regulation of programmed cell death (PCD). The aim of our study was to determine whether mRNA levels of Mdm-2, Bcl-2, Bcl-x(L), Bad, and Bax are independent prognostic parameters for outcome. Transcript levels were analyzed by real-time quantitative RT-PCR in 232 samples collected either at diagnosis or following induction chemotherapy (ICT). Multivariate COX regression analysis adjusted for chemotherapy protocol, de novo vs secondary AML, and de novo vs relapsed AML indicated: (1) At diagnosis, high expression of Bad (P = 0.015) and even more so high Bax and Bad levels (P = 0.018) predicted adverse outcome, regardless of the response to ICT. In patients who subsequently failed to enter complete remission (CR), high levels of Bad, Bax and Bax high/Bad high were associated with an increased relative risk (RR) to die from tumor (RR = 5.0 for Bad, 3.4 for Bax and 6.14 for Bax high/Bad high). (2) Following ICT, high expression of Bax (P= 0.005) and high Bcl-2/Bax ratios (P = 0.004) were independent predictors of unfavorable outcome, regardless of response to ICT. We conclude that high levels of Bax and Bad correlate with poor outcome, particularly in patients who do not enter CR and may serve as prognostic markers in AML.

Chemotherapy for mobilisation of Ph-negative progenitor cells from patients with CML: impact of different mobilisation regimens.

Mobilised peripheral blood stem cells are widely used for autografting in patients with chronic myeloid leukaemia (CML) and it is generally thought that a high proportion of Ph-negative progenitor cells in the graft is desirable. We report here the results of 91 stem cell mobilisations performed with various chemotherapy regimens followed by G-CSF. We show that mobilisation of Ph-negative cells is possible after diagnosis as well as in advanced stages of the disease. The yield of Ph-negative cells is highly dependent on the chemotherapy regimen: while the combination of idarubicin and cytarabin for 3-5 days (IC3-5) mobilised Ph-negative cells in most patients, high-dose cyclophosphamide was ineffective. Mobilisation of Ph-negative progenitor cells after IC3 was at least as effective as after IC5; however, less apheresis sessions were required, and toxicity was much reduced after IC3. Compared to historical controls, IC was equally effective as the widely used ICE/miniICE (idarubicin, cytarabin, etoposide) protocol. No correlation was found between graft quality and the cytogenetic response to subsequent treatment with interferon-alpha. We conclude that IC3 is an effective and well-tolerated regimen for mobilising Ph-negative cells that compares well with more aggressive approaches such as IC5 and ICE/miniICE.

Absolute levels of MDR-1, MRP, and BCL-2 MRNA and tumor remission in acute leukemia.

Mononuclear cells prepared from peripheral blood or bone marrow of 119 AML and 28 ALL patients prior and following therapy were analyzed for absolute transcript levels of the chemoresistance genes mdr-1 and MRP, and the proto-oncogene bcl-2, by validated contamination-protected quantitative RT-PCR. In newly diagnosed AML mainly tumors of the granulocytic lineage (FAB M1-M2) expressed increased mdr-1 mRNA amounts. The MRP gene was expressed in all investigated samples without relation to a particular FAB class. High initial expression of both genes did not confer a poor prognosis even at high number of CD34+ cells. Data compared prior to and after therapy start (paired samples) revealed that AML patients who did not respond to therapy (NR) expressed increased levels of mdr-1 mRNA, as well as MRP and bcl-2 cDNA normalized to GAPDH reference transcripts, when compared to patients achieving complete remission (CR; p = 0.003, 0.008 and 0.0005, respectively). In ALL-NR the mdr-1 and bcl-2 genes were entirely more active after induction chemotherapy. Arbitrary cut-off values were established in order to delimit pathological from non-pathological gene expression. 59% of studied AML and 33% of ALL-NR exceeded the arbitrary values (mdr-1: > 2 amol/microgram RNA, MRP: > 10 zmol/amol GAPDH, bcl-2: > 5 zmol/amol GAPDH) for one and 11% of AML-NR for two parameters. Only 17% of the AML-CR and none of the ALL-CR group were above these limits. The results indicate that high individual activity of usually one, rarely two of the investigated genes might be associated with poor clinical outcome in treated acute leukemia.

HCG secretion by peripheral mononuclear cells during pregnancy.

Peripheral mononuclear cells (MNC) in culture release a biologically active hCG. This effect is detectable during pregnancy with a maximum between the 9th and 16th wk. Peripheral MNC already secrete hCG between the 7th and 11th d after embryo transfer. The secretion of hCG is activated by the PKC-activator TPA. TPA induces hCG release into the medium, thus causing a decrease in intracellular hCG content. In contrast, db-cAMP inhibites hCG secretion into the medium. Protein synthesis inhibitors of transcription and translation suppress the production and secretion of hCG. Peripheral natural killer (NK) cells (CD56+/CD16+) and monocytes (CD14+) show the highest secretion rates. IL-1 beta, IL-4, IL-6, IL-10, TNF alpha, and GM-CSF stimulate, whereas IL-2 and INF gamma inhibit, the hCG secretion of mononuclear cells. Flow cytometric experiments with hCG antibody demonstrate a binding of hCG on the surface of monocytes more than lymphocytes. The binding capacity is improved during pregnancy. Different hCG bands are shown in the Western blot analysis. We could confirm the mRNA of beta hCG and alpha CG are in MNC as well in the placental control. Peripheral MNC, first and foremost NK cells and monocytes, produce and secrete hCG during pregnancy, which play an important role for the corpus luteum rescue during the early gestational age and possibly for the immunotolerance.

T-cell depletion and haematological side effects of two cytotoxic monoclonal antibodies.

We investigated two cytotoxic monoclonal antibodies of BL-series (BL-IIIB4 and BL-IIG2) according to T-lymphocyte depletion from bone marrow. Both antibodies work together with human complement similar Campath-1, which was tested parallelly. The extent of T-cell depletion is about 95% for all three antibodies. On the other hand the haemopoietic side effects tested by CFU-GM recovery and LTBMC are for the BL-antibodies not as strong as for Campath-1, especially in view of LTBMC. T-cell regeneration could be shown in long term cultures. Our results indicate a possible suitability of the two investigated antibodies for T-cell depletion of bone marrow.

Immune recovery following bone marrow transplantation.

83 patients undergoing allogeneic or autologous BMT because of haematologic malignancies have been studied before and after transplantation at different intervals. The determinations consisted of lymphocyte counts, E-rosetting, lymphoblastic response, evaluation of serum immunoglobulin levels, skin testing, and in a smaller part of the patients surface marker studies using monoclonal antibodies of the BL-series. At first after BMT the lymphocyte and T cell counts went to normal between 4-18 weeks post transplant, about 4 weeks earlier in autologous than in allogeneic BMT. T suppressor cells showed an early increase compared to T helper cells which normalized much slower about 6 months after BMT. Lymphoblastic responses, however, tended to normal not before the second half of the first year both in autologous and allogeneic transplantation. Skin test reactivity became normal during the 2nd and 3rd year posttransplant, which was more complete in autologous than in allogeneic BMT. The IgG and IgM levels were depressed for half a year and IgA levels for 2 years. The most striking aspect was the multiphase course of lymphoblastic response in every individual patient. We suggest this to be the expression of sequential differentiation of donor lymphocytes.

[Results of therapy of 100 patients with acute leukemia--a retrospective unicenter 5-year analysis]. / Therapieergebnisse bei 100 Patienten mit akuter Leukämie--eine retrospektive unizentrische 5-Jahres-Analyse.

The results of therapy in 100 patients who newly fell ill (68 AML, 32 ALL) with acute leukaemia were evaluated (1981 to 1985). The 5-year-survival chance of all patients is 15% for AML, 18% for ALL, first of all it is depending on the degree of remission obtained. The CR rate is nearly 43% (AML) and 66% (ALL), respectively, shows a dependence upon age and is impaired above all by a high early death rate (supportive therapy). With increasing aggressiveness the results of the remission induction therapy improve, as it becomes clear in a comparison with an evaluation of patients 1965-1980 (CR: 15-32%). Also in the postremission therapy the results of intensive forms of therapy are more favourable: 4 years recurrence-free survival after CR in autologous bone marrow transplantation 50%, in allogenic bone marrow transplantation 40%, in cyclic chemotherapy 17%, in oral permanent therapy 0%. Starting from these findings the present conception of the therapy of acute leukaemias is discussed in connection with the literature.

[Testing of acute myeloid leukemia of humans using monoclonal antibodies BL-DR, BL-M/G, BL-T2 and BL-Ig-L/1]. / Testung von akuten myeloischen Leukämien des Menschen mittels der monoklonalen Antikörper BL-DR, BL-M/G, BL-T2 und BL-Ig-L/1.

The leukemic blasts of 22 patients with acute myelocytic and myelomonocytic leukemia were tested by indirect immunofluorescence with the monoclonal antibodies BL-DR (directed against HLA-DR-antigens), BL-M/G (react with both granulocytes and monocytes), BL-T2 (pan-T-lymphocyte-antibody CD 5), and BL-Ig-L/1 (anti-light-chains-antibody). The leukemic blasts showed no crossreaction with BL-DR and BL-Ig-L/1. Both the antibodies BL-DR and BL-M/G reacted mainly with the acute myelomonocytic leukemias FAB M4 and only seldom with the acute myelocytic leukemias FAB M1, M2, and M3. The antibodies BL-DR and BL-M/G are able to confirm the diagnosis of acute myelomonocytic leukemia and to classify some previously unclassifiable leukemias.

[Use of monoclonal antibodies in the diagnosis of acute leukemia]. / Einsatz der monoklonalen Antikörper in der Diagnostik der akuten Leukämien.

Immunological analysis has demonstrated the heterogeneity of acute lymphoblastic leukemia (ALL). The use of classical immunological methods and monoclonal antibodies against human lymphoid cell surface antigens have made it possible to subdivide ALL into five major subclasses: null ALL, common ALL, T-ALL, pre-B-ALL and B-ALL. We have studied in this report 8 cases of adult ALL diagnosed on morphological and cytochemical criteria. Some conventional markers and a panel of monoclonal antibodies have been used to analyse the differentiation of those patients' blast cells. In a second part we report two cases in which myeloid and lymphoid cell markers were observed simultaneously when 22 patients with acute myeloid leukemia were phenotyped according to the French-American-British (FAB) classification system.

In vivo DNP tolerance induction by DNP-ficoll.

The kinetics of tolerance induction by administration of two different doses of DNP78-Ficoll on the anti-DNP IgM immune response in AJ mice was studied. The tolerogenic effects of 100 microgram of DNP78-Ficoll begin earlier and last longer than those of 10 microgram of DNP78-Ficoll as determined by the development of direct anti-DNP plaque-forming cells in the spleen and anti-DNP antibody titers in serum. In cell-transfer experiment, DNP tolerance was unstable. Transfer experiments did not produce detectable tolerogenic factors in serum. Antigen-antibody complexes, that tend to produce local suppressor effects, are discussed as mediators of tolerance.

[Additive suppressive effect of Freund's complete adjuvant on the specific inhibition of anti-DNP immunoreaction (author's transl)]. / Die additive suppressive Wirkung von komplettem Freundschen Adjuvans bei der spezifischen Hemmung der Anti-DNP-Ficoll-Immunreaktion.

Injection of DNP 38-Ficoll in 0,9 per cent saline causes a specific suppression of the secondary anti-DNP reaction in AJ-mice to the same antigen. This suppression is amplified by injection of the tolerogen in Freund's complete adjuvant (FCA) or by simultaneous injection of the tolerogen and FCA. --This additive suppressive effect of FCA is distinctly better if there are used small doses of tolerogen. Possible mechanisms interpreting these results are discussed.