ALBERT

Description: A woman with lymphoblastic lymphoma was treated with combination chemotherapy. She subsequently became febrile while granulocytopenic and was given unirradiated granulocyte transfusions from normal, unrelated donors. She recovered, but 12 days later noted the onset of progressive skin rash, hepatic dysfunction, diarrhea and pancytopenia and, 22 days after her last granulocyte transfusion, died of gram negative septicemia. Histologic examination of multiple tissues including the skin, liver, and intestinal tract showed changes characteristic of acute graft-versus-hose disease (GVHD). Y-chromatin analysis of the patient's peripheral blood just before death indicated the presence of male cells. HLA typing of lymphocytes and skin fibroblasts from the patient and lymphocytes from the family and granulocyte donors was also consistent with engraftment of cells from one of the male granulocyte donors. This donor most likely was homozygous for one of the patient's halotypes, perhaps facilitating engraftment of his cells and subsequent development of transfusion- induced acute GVHD. Until more precise guidelines can be established, we recommend that all cellular blood products given to patients receiving intensive chemotherapy be irradiated with 1500 rad.

Description: We hypothesized that after allogeneic hematopoietic stem cell transplant (HSCT), GvHD-affected tissues harbor expanded “immunodominant” T-cell clones, and characterization of these clones can be used to develop markers of disease. A multiplex PCR was used to detect T-cell receptor variable beta (VB) chain rearrangements in target tissue. Molecular analysis of the amplified VB CDR3 sequences allowed for identification and quantitation of putative disease-associated “clonotypes” and for the development of clone-specific PCR. We studied 5 HSCT patients for the presence of signature clonotypes in 10 skin biopsies taken during diagnostic GvHD work-up. Size distribution analysis of VB PCR products showed a skewed peak pattern in 9 biopsies; immunodominant clones (per definition frequency ≥30%) were detected in 6/7 biopsies with histologically confirmed GvHD, consistent with the presence of expanded clonotypes and the oligoclonal nature of the tissue-specific alloresponse. Immunodominant clones were also found in 2 of 3 biopsies not diagnostic for GvHD but obtained based on strong clinical suspicion, raising the possibility that they were associated with early evolving GvHD not distinguishable by histology. For example, when serial skin biopsies were analyzed, a GvHD-positive post-transplant d63 biopsy contained an immunodominant clone (frequency 60%), which was also detected in a subsequent biopsy positive for GvHD (frequency 33%). Similar results were seen in another patient, in whom serial biopsies taken on d214 (not diagnostic) and d217 (GvHD-positive) showed an identical immunodominant clone, not present in a d13 GVHD-negative biopsy. This finding suggests that the d214 biopsy might have contained early GvHD that was not detectable morphologically. In a patient who rejected an initial MUD graft (Tx 1) and then received a MUD SCT (Tx 2) from a different, unrelated donor, immunodominant clones were identified in GvHD-positive biopsies following each transplant, that were distinct for each graft. To examine whether immunodominant clonotypes derived from biopsies could be used as markers of disease, clonotypic PCR was developed for an immunodominant biopsy-derived clonotype for each transplant. The Tx 1 clonotype was detected in blood and skin following Tx 1, but not in tissue or blood taken after Tx 2. Specificity and correct size of the clonotypic PCR product were confirmed by both Genescan analysis and sequencing. Clonotypic PCR designed for an immunodominant clonotype from the Tx 2 donor detected the putative allospecific clonotype in serial samples after the second engraftment. Neither clonotype could be found in either donor, indicating that the disease-associated clones expanded to detectable levels following transplant. These results indicate that clonotypic PCR can distinguish distinct GvHD-associated clonotypes from different donors in both blood and tissue following transplant. Monitoring of the relative frequency of disease-associated clones in recipient blood indicated a significant peripheral expansion of disease-associated clones at the time of active GvHD. Our results demonstrate an efficient method for identification of disease-associated clonotypic markers, which can be used to aid diagnosis and monitoring of GvHD.

Description: A woman with lymphoblastic lymphoma was treated with combination chemotherapy. She subsequently became febrile while granulocytopenic and was given unirradiated granulocyte transfusions from normal, unrelated donors. She recovered, but 12 days later noted the onset of progressive skin rash, hepatic dysfunction, diarrhea and pancytopenia and, 22 days after her last granulocyte transfusion, died of gram negative septicemia. Histologic examination of multiple tissues including the skin, liver, and intestinal tract showed changes characteristic of acute graft-versus-hose disease (GVHD). Y-chromatin analysis of the patient's peripheral blood just before death indicated the presence of male cells. HLA typing of lymphocytes and skin fibroblasts from the patient and lymphocytes from the family and granulocyte donors was also consistent with engraftment of cells from one of the male granulocyte donors. This donor most likely was homozygous for one of the patient's halotypes, perhaps facilitating engraftment of his cells and subsequent development of transfusion- induced acute GVHD. Until more precise guidelines can be established, we recommend that all cellular blood products given to patients receiving intensive chemotherapy be irradiated with 1500 rad.

Description: LBH589 is a novel HDAC inhibitor which activates p21 and inhibits proliferation and induces apoptosis in tumor cell lines at nanomolar concentrations. In this study, LBH589 was administered IV as a 30-minute infusion on days 1–7 of a 21 day cycle. Fourteen pts. (median 61 years [range 42–87], performance status 0 (2 pts) or 1 (12 pts), de novo AML (2 pts), relapsed /refractory AML (10 pts), refractory ALL (1 pt), MDS (RAEB) (1 pt) have been treated at dose levels (mg/m2): 4.8 (3 pts), 7.2 (3 pts), 9.0 (1 pt), 11.5 (2 pts), 14.0 (5 pts). Four DLT’s (G3 QTcF prolongation) have been observed − 3 at 14.0 mg/m2 and one at 11.5 mg/m2 (a total of 357 ECG’s were performed during the study). One patient treated at 14.0 mg/m2 died of pulmonary hemmorhage resulting from sepsis while on study. Treatment with LBH589 as well as the patient’s underlying disease (MDS) were considered a contributing factors to the sepsis. A white cell differentiation syndrome (which was sucessfully treated with high-dose steroids) was observed in one patient with refractory AML treated at 14.0 mg/m2. Other LBH589-related toxicities included: Grade 1 ST-T wave abnormality, palpitations, hypokalemia; Grade 2 diarrhea, nausea, vomiting, loss of appetite, headache, atrial fibrillation, and QT prolongation. In 9 of 12 patients (2 patients were aleukemic), treatment with LBH589 resulted in reductions in peripheral blasts, WBC and platelets during treatment. Counts rebounded following the 7-day treatment period (generally by day 15). Bone marrow blast counts generally continued to increase during treatment. The levels of histone acetylation in bone marrow and peripheral blood cells were measured using quantitative flow cytometry and antibodies against histones H2B and H3. The median acetylation of histones H2B and H3 in CD34+ cells increased on therapy from 4463 to 17185 and from 11540 to 66224, respectively. Despite this increase in histone acetylation a significant change in apoptosis (measured by annexin V or mitochondrial potential) or proliferation (measured by BrdU incorporation) in CD34+ cells was not demonstrated. However, peripheral blood lymphocytes showed significant increase in apoptosis on day 7 as compared with the levels of apoptosis before therapy. PK samples were collected on days 1 and 7 of cycle 1 and analyzed using a noncompartmental analysis. Plasma LBH589 concentrations were determined using HPLC/MS/MS assay. AUC increased proportionally with dose and mean AUCs (0–24h) were 139.47, 131.46, 189.12, and 344.28 ng.h/mL, respectively, for 4.8, 7.2, 9.0, and 14.0 mg/M2 dose levels on Day 1. Terminal half-life was approximately 11 hrs. LBH589 given IV appears well tolerated at doses below 11.5 mg/m2 with consistent transient antileukemic and PD effects.

Description: Physiologic concentrations of human plasma very low density lipoproteins inhibit the DNA synthesis of lymphocytes stimulated by allogeneic cells or lectins. In this report we have compared the effects of isolated lipoproteins [very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)] and lipoprotein-depleted plasma (LDP) on DNA synthesis by phytohemagglutinin-stimulated human lymphocytes. The relative potency for the inhibition of lymphocyte proliferation was VLDL greater than LDL greater than HDL greater than LDP. Fifty percent inhibition of DNA synthesis was observed at a VLDL protein concentration of 1.5--2.0 microgram/ml. We have further demonstrated the presence of specific receptors for VLDL on human lymphocytes. Native VLDL was more effective than LDL in competing for 125I-VLDL binding sites. Subsequent to binding to lymphocytes, 125I-VLDL was internalized and degraded to acid- soluble products. Based on a Scatchard analysis of VLDL binding at 4 degrees C, the number of VLDL receptors per lymphocyte was estimated at 28,000 +/- 1300. Based on an estimated mean binding affinity for the VLDL receptor complex at half saturation of approximately 8.8 X 10(7) liter/mole, it is estimated that 91% of lymphocyte VLDL receptors are occupied at physiologic VLDL concentrations in blood. Although the immune regulatory role of plasma lipoproteins is uncertain, we suggest tha VLDL and LDL-In may maintain circulating blood lymphocytes in a nonproliferative state via their respective cell receptor mechanisms.

Description: Calf kidney has been used as a tissue source for the isolation of cobalamin analogues, which are defined here as cobalt-containing compounds of distinctive chromatographic behavior that are extractable from tissues by methods conventionally used to extract cobalamin and which, in radioisotope dilution assays, are more active with R-protein as binder than intrinsic factor and are relatively less active in microbiologic assays. Preparatory methods employed reverse affinity chromatography or a series of chemical extractions for the isolation of corrin followed by Dowex-50 chromatography. An analogue-containing fraction (peak 2) was eluted by acetate buffers between pH 4 and 5. This material was shown to contain cobalt, to migrate differently than the four cobalamins in Dowex-50 and paper chromatography, and to display a pattern of properties that is compatible with the above definition of cobalamin analogues. These analogues stimulated crude preparations of N5-methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13) from Escherichia coli and rat liver at far lower concentrations (1–40 nmol/L) than the major cobalamins. No evidence of enzymatic conversion of cobalamin to analogue could be demonstrated.

Description: Invasive aspergillosis is still a life-threatening complication, in particular in patients after allogeneic hematopoietic stem cell transplantation (HSCT). Whereas prolonged neutropenia is a well established risk factor for invasive fungal disease, there is a growing body of evidence that T-cells also play an important role in the immunological response to Aspergillus species. Since invasive aspergillosis often occurs during the phase of postengraftment, which is characterized by impaired cell-mediated immunity, Aspergillus-specific T-cells could be a potential therapeutic target in these patients. We therefore analyzed in a first step the response of T-cells to several potential antigens of Aspergillus fumigatus by means of 3H-thymidine incorporation assay. In order to generate Aspergillus-specific T-cells, the antigens with the highest proliferation indices (EC-SAB and 90 kDa catalase) were used to stimulate 1.0 x 108 mononuclear cells from healthy donors. The activated T-cells were isolated on the following day using the IFN-γ secretion assay (Miltenyi Biotec, Germany) and then expanded for 14 days. Intracellular cytokine analysis of EC-SAB generated cell lines (n=7) revealed a significant IFN-γ secretion by 13.6%±2.3 of CD4+ cells (seven out of seven tested cell lines) and an Aspergillus-specific IL-2 secretion by 6.5%±1.9 of CD4+ cells (three out of three tested cell lines), which supports the TH1 response of the generated cells to Aspergillus antigen. In contrast to EC-SAB generated T-cell populations, all three cell lines which were generated with 90 kDa catalase were not informative. Further analysis showed that restimulation with EC-SAB induced a strong proliferation of EC-SAB generated T-cell populations (all three populations tested), whereas alloreactivity was unaffected. The number of these cells could be expanded within 14 days up to 20fold using OKT-3, IL-2 and feeder cells. Currently, we investigate the impact of these Aspergillus-specific cell populations in the defense to different species of Aspergillus. Our preliminary results suggest that Aspergillus-specific T-cells could be an interesting option in prophylaxis and therapy of invasive aspergillosis in patients undergoing HSCT.

Description: T-cell acute lymphoblastic leukemia (T-ALL) accounts for 25% of adult ALL and is characterised by specific clinical and biologic features such as phenotype with early (cyCD3+,CD7+) (E-T), thymic (CD1a+) ) (Thy-T) and mature T-ALL (M-T) (sCD3+). The formerly poor prognosis of T-ALL was more recently improved in some studies albeit not in all, even not in childhood ALL. Patients: To improve outcome of T-ALL the German Multicenter Study Group for Adult ALL initiated two consecutive studies with subtype adapted therapies. 503 T-ALL pts were recruited between 4/93 and 10/03. The median age was 30 (15–55)yrs, 75% were male, 66% had mediastinal tumor (MedTu), 24% WBC 〉 100.000 and 7% CNS involvement with subtypes as follows: 53% Thy-T, 26% E-T and 21% M-T. Study Design: In Study 05/93 all T-ALL pts were treated uniformly with 8drug induction incl. prophylactic CNS (24 Gy) (CNSRAD) and proph. mediastinal (24 Gy) irradiation (MEDRAD) followed by 7x consolidation (HDAC/MITOX, HDMTX/ASP, reinduction, 2xVM26/AC, 2xCYCLO/AC) and maintenance (6MP/MTX). In Study 06/99 treatment of T-ALL was risk adapted with a shortened, intensified 8drug induction (CNSRAD in all but MEDRAD only in pts with residual MedTu after induction) followed by consolidation I (HDAC/HDMTX/VP16). Thy-T was then treated as standard risk with 6x consolidation (3xHDMTX/ASP,reinduction,VM26/AC,CYCLO/AC). E-T and M-T were considered as high risk and scheduled for stem cell transplantation (SCT) in CR1. Results: In Study 05/93 the CR rate in 291 pts was 89% (94%, 73% and 90% for Thy-T, E-T and M-T; p=.0006) and even 97% for Thy-T in adolescents (15–25 yrs) . Overall 4% failed to achieve CR and 7% died in induction. The probability of continuous CR (CCR) at 5 yrs was overall 53% and 64% for Thy-T, but only 30% (p

Description: Despite the major peak of ALL in childhood the incidence also increases in the elderly beyond 50 yrs. Outcome of these elderly ALL pts is very poor. In a survey including 12 studies with 370 pts (50–88 yrs) the mean CR rate was 50% and the rate of continuous CR (CCR) 13%. Also in a first prospective pilot study of the German Multicenter Study Group for Adult ALL (GMALL) for elderly ALL pts 〉 65 yrs with a moderate intensive chemotherapy regimen (Blood, 96(11): 3104a, 2000) the results in 94 pts were poor with a CR rate of 48% and a survival (OS) 55 yrs (age limit for allo SCT) according to status for 1) Ph/BCR-ABL, 2) CD20 expression and 3) a separate protocol for mature B-ALL. For 1) and 2) chemotherapy was similar to the pilot study (induction I,II consol. I–V with 2xIDMTX/MP,2xVM26/AC and reinduction). Out of a total of 824 pts recruited for the GMALL 06/99 109 were older than 55 yrs. 1) Imatinib in Ph/BCR-ABL+ ALL: In a prospective randomized trial pts received a 4 wk induction with 600 mg/d Imatinib (ImInd) compared to chemotherapy without Imatinib (ChInd). 36 pts (median age 67y) were evaluable (18 ImInd; 18 ChInd). The CR rate was higher for the Imatinib arm with 93% compared to only 44% with chemotherapy (p=.003). 3 of the 4 failure pts in the ChInd arm achieved CR after cross-over to ImInd. There were more non-hematologic severe (grade III/IV) adverse events during ChInd (pneumonia N=6 sepsis 2, enteritis 2, hepatotoxicity 1) versus ImInd (N=0). After induction both arms received the consolidation cycles parallel with Imatinib 600 mg/d for 1 yr. After a median follow-up of 4.3 mo (0.5–20+) 21 pts are in CCR and 6 relapsed. 7 pts died in CR. The OS at 18 mo is 47%. 3 pts (11%) achieved molecular remissions. 2) Rituximab in CD20+ ALL: In the 2nd study pts with CD20+ B-prec. ALL (Ph/BCR-ABL neg) received 375 mg rituximab before each cycle (induction I, II, consolidations). The small group of CD20neg ALL received the similar chemotherapy regimen without rituximab. 26 pts are evaluable (19 with rituximab and 7 with CD20 neg B-prec. or T-ALL). The median age is 66 (55–79) yrs. The CR rate in CD20+ pts is 63% and the OS after 1 yr is 54%. No pts died in CR so far. 3) Rituximab in mature B-ALL, Burkitt or other high-grade NHL: Elderly pts 〉 55 yrs with these diseases had a poor outcome with the former protocol B-NHL90 (Blood100(11):159a, 2002). The CR-rate in 45 pts was 71% and the OS 39% at 6 yrs. Therefore in the subsequent trial B-NHL2002 pts with mature B-ALL/B-NHL 〉55 yrs (86% CD20 pos) received a total of 8 cycles rituximab (375 mg), 6x before each chemotherapy cycle (day −1) and 2x rituximab only. In 26 evaluable pts the CR-rate increased to 81% and the OS at 1.5 yr is 84% (p=.03 compared to study B-NHL90). Out of 21 CR pts 19 are in CCR, 1 relapsed and 1 went off-study. Conclusion: In elderly ALL pts the application of Imatinib in Ph/BCR-ABLpos or rituximab in CD20+ pts led to a stubstantially improved antileukemic activity with high CR and low failure rates. The risk of infections remains a major problem and improved supportive measures are needed. Hopefully this risk stratified approach with targeted therapies will translate into improved long-term outcome in this age category (partly supported by Novartis Pharma and Hoffmann-La-Roche)

Description: Calf kidney has been used as a tissue source for the isolation of cobalamin analogues, which are defined here as cobalt-containing compounds of distinctive chromatographic behavior that are extractable from tissues by methods conventionally used to extract cobalamin and which, in radioisotope dilution assays, are more active with R-protein as binder than intrinsic factor and are relatively less active in microbiologic assays. Preparatory methods employed reverse affinity chromatography or a series of chemical extractions for the isolation of corrin followed by Dowex-50 chromatography. An analogue-containing fraction (peak 2) was eluted by acetate buffers between pH 4 and 5. This material was shown to contain cobalt, to migrate differently than the four cobalamins in Dowex-50 and paper chromatography, and to display a pattern of properties that is compatible with the above definition of cobalamin analogues. These analogues stimulated crude preparations of N5-methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13) from Escherichia coli and rat liver at far lower concentrations (1–40 nmol/L) than the major cobalamins. No evidence of enzymatic conversion of cobalamin to analogue could be demonstrated.