bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the ZAP70+ and ZAP70− B-CLL Kinome: Higher Kinase Activity in ZAP70+ B-CLL Cells.
Authors: Wang Miao , Rosati Stephano , Parikh Kaushal , Peppelenbosch Maikel , Daenen Simon , Kluin P.M. , Visser Lydia
Published: October 2019

B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with a highly variable clinical course. The expression of ZAP70 can serve as a novel prognostic marker in B-CLL, but the function of ZAP70 in the disease is still unknown. A high throughput analysis of protein kinases allows a comprehensive detection of signal transduction events in CLL. The goal of this study was to detect the activity of kinases in B-CLL cells and compare the kinase activity of (IgM positive) B-CLL cells immediately after stimulation through IgM. We compared 2 cases of ZAP70+ and 2 cases of ZAP70− untreated B-CLL patients. Tumor cells were isolated from lymph node cell suspensions, purified by negative selection (> 95% purity), and cross linked with biotin labeled anti-IgM F(ab)2 (10 μ g/mL) followed by avidin (10 μ g/mL) for 10 minutes at 37°C. Whole cell lysates in combination with 32 P-ATP were overlaid on a kinomics chip, which consists of 1024 peptides in triplicate (including 48 peptides as internal controls) and can detect the activity of 207 different kinases.

We found significant differences of the activity of 74 kinases between ZAP70+ and ZAP70− cases, with a higher phosphorylation of 93 peptides in the ZAP-70+ group. Phosphorylation of ZAP70 by kinase LCK was up regulated with a factor 2.7 in the ZAP-70+ B-CLL group. The kinases SYK, LCK, Fyn, HPK1, CAMK2A, PRKAR1A and PKC all showed a higher activity in the ZAP70+ group. These kinases are mainly involved in the B cell receptor pathway and indicate that ZAP70+ B-CLL cells are more activated than the negative group. The kinases CDC2, CDK2, PLK, and ABL also showed a higher activity in the ZAP70+ B-CLL cases, indicating a higher proliferation rate. PTK6 and LIMK1 which also had a higher activity in the ZAP70+ group are oncogenic kinases and play a fundamental role in transformation and migration.

Crosslinking of the BCR via IgM resulted in changes in the activity of 73 kinases, 55 showing a lower activity. Since ZAP70 expression can be induced through activation in normal B-cells, it is possible that the already highly activated status of the ZAP70+ cells causes a fast dampening after IgM stimulation. In the ZAP70− group only a minimal change was detected upon IgM cross linking. After 10 minutes stimulation with anti-IgM, the activity of ZAP70 itself as assessed by alterations of the phosphorylation status of its substrate HIP-55 was down regulated in exclusively the ZAP70+ cases. HIP55 possibly connects ZAP70 with both the BCR and apoptosis pathways.

In summary, ZAP70 is not only a prognosis marker of B-CLL, but might also play a key role in survival, proliferation, activation and transformation of B-CLL cells.
Introduction of Constitutivelly Active Akt in Primary CLL B-Cells Results in Upregulation of Mcl-1, Bcl-XL and Cyclin D3 and Promotes Leukemic Cell Survival.
Authors: Longo Pablo , Gobessi Stefania , Laurenti Luca , Sica Simona , Leone Giuseppe , Efremov Dimitar G.
Published: October 2019

The PI3K/Akt and Raf/MEK/ERK pathways are key regulators of various cellular responses, including proliferation, survival, differentiation, migration and malignant transformation. These pathways are activated in chronic lymphocytic leukemia B-cells by a number of survival or growth stimulatory signals, such as immobilized anti-IgM antibodies, interleukin-4, phorbol-ester, CXCL12, or stimulatory CpG oligonucleotides. Moreover, enhanced activation of Akt has been implicated in the pathogenesis of the CLL-like disorder that develops in mice transgenic for the TCL1 oncogene. To further delineate the relative contribution of the PI3K/Akt and Raf/MEK/ERK pathways in regulating leukemic cell growth and survival, we introduced constitutively active Akt or constitutively active MEK2 in primary CLL B-cells by nucleofection. Expression of constitutively active Akt consistently promoted survival, as evidenced by a higher percentage of Annexin V/PI negative cells after 48 hours in culture (median 52%) compared to samples transfected with control vector (median 31%). Immunoblot analysis of several important antiapoptotic proteins revealed that enforced activation of Akt upregulates Mcl-1 and Bcl-xL, whereas no changes were observed in the levels of Bcl-2. Expression of constitutively active Akt also induced an increase in size and granularity of the leukemic cells, indicating increased metabolic activity. These changes were associated with significant induction of cyclin D3, indicating that activation of Akt is required for both leukemic cell survival and cell cycle progression. In contrast, introduction of constitutively active MEK2 induced sustained activation of ERK, but showed only a modest increase in the percentage of viable CLL B-cells (median 36%) and no significant changes in the levels of any of the investigated antiapoptotic proteins. These experiments provide direct evidence that sustained activation of Akt promotes leukemic cell survival and upregulates Mcl-1, an antiapoptotic protein that has been associated with resistance to chemotherapy in patients with CLL.
CD21 Expression Is Inversely Correlated with the Ability of CLL Cells To Induce Tyrosine Phosphorylation in Response to Anti-IgM.
Authors: Pepper Chris , Alghazal Suhair , Brennan Paul , Rowntree Clare , Lin Thet Thet , Pratt Guy , Fegan Chris
Published: October 2019

B-cell chronic lymphocytic leukaemia (CLL) cells have a differential capacity to signal through the B-cell receptor (BCR) following ligation with anti-IgM. This difference in signaling capacity has been suggested to contribute to the clinical disparity between the two subsets. However, as there appears to be no obvious correlation between response to IgM ligation and surface IgM expression, CD20 expression or CD79b expression it may be that the differential signaling response in these two distinct subsets is not caused by the BCR itself. In this study we investigated the expression of the BCR co-receptor complex (CD19, CD21 and CD81) in a cohort of CLL patients (n = 60) in order to determine whether expression of the co-receptor was associated with known CLL prognostic markers; IgVH gene mutational status, ZAP-70 and CD38 expression. In addition, we examined the relationship between co-receptor expression and the capacity of CLL cells to respond to anti-IgM cross-linking of the BCR. CLL samples showed significantly lower expression of both CD19 and CD21 when compared to normal B-lymphocytes (P <0.0001 and P<0.0001 respectively). In addition, samples derived from patients with unmutated IgVH genes showed significantly lower expression of CD19 and CD21 but not CD81 when compared to samples derived from patients with mutated VH genes (P = 0.04, P = 0.005 and P = 0.5 respectively). In contrast, none of the co-receptor molecules (CD19, CD21, CD81) were associated with CD38 expression (P = 0.82, P = 0.6, P = 0.64 respectively) or ZAP-70 expression (P = 0.2, P = 0.13 and P = 0.4 respectively). We next compared CD21 expression with levels of tyrosine phosphorylation (pre- and post-IgM ligation of the BCR). The results showed that there was no correlation between CD21 expression with either basal or IgM-stimulated tyrosine phosphorylation (P = 0.84 and P = 0.24 respectively). However, there was a significant inverse correlation with the percentage change in tyrosine phosphorylation following BCR cross-linking (P = 0.01). These data suggest that CLL cells with higher CD21 expression have a diminished magnitude of response to BCR ligation supporting the hypothesis that CLL cells with higher CD21 expression undergo receptor densitisation. Conversely, patients with lower CD21 expression may avoid receptor densitisation and consequently have a higher capacity for signaling following ligation of the BCR. Taken together, our data indicate a role for the BCR co-receptor in the signaling capacity of CLL cells and hence in the clinical course of the disease.
Humoral Immune Responses Against the Oncofetal-Antigen/Immature Laminin Receptor (OFA/iLR) in Patients with Chronic Lymphocytic Leukemia.
Authors: Friedrichs Birte , Siegel Sandra , Schmitz Norbert , Zeis Matthias
Published: October 2019

The 37 kD oncofetal antigen/immature laminin receptor (OFA/iLR) is a strongly overexpressed tumor antigen on malignant cells of numerous tumor entities including several hematological malignancies but it is not present on the cell surface of normal differentiated tissues. OFA/iLR is widely expressed on chronic lymphocytic leukemia (CLL) cells while absent on different normal B-cell subsets. The aim of this study was to evaluate and characterize humoral immune responses towards OFA/iLR in patients with CLL. 38 healthy individuals and 68 untreated patients with chronic lymphocytic leukemia were analysed for the presence of anti-OFA/iLR-antibodies. Sera were screened in an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) and IgM anti-OFA/iLR-antibodies. Detectable IgG or IgM responses to OFA protein were present in 32 of 68 patients (47,0%) but only in 4 of 38 (10,5%) healthy donors. IgG subtype analysis revealed a predominant IgG1 and IgG3 response. Utilizing 42 peptides deduced from the complete OFA/iLR amino acid sequence, 24 (35,3%) samples with significant anti-OFA/iLR IgG and/or IgM titers were also reactive with at least one OFA/iLR epitope. Patient-derived anti-OFA/iLR antibodies were capable of recognizing and selectively killing OFA/iLR expressing CLL cells in complement-mediated and antibody-dependent cellular cytotoxicity (ADCC) assays. Kaplan-Meier plots for progression-free survival (PFS) revealed a tendendy for longer PFS in patients with detectable humoral responses compared patients with negative ELISA (p=0,0165).

In addition, sera from eight CLL patients who had undergone allogeneic hematopoetic stem cell transplantion (allo-HSCT) were analysed. Six out of eight patients showed high values for IgG or IgM anti-OFA/iLR-antibodies after allo-HSCT as long as remaining in complete remission suggesting a potential role of anti-OFA/iLR-directed Graft-versus-Leukemia effects. For the first time, these data suggest that anti-OFA/iLR antibodies might be involved in the immunological control of OFA/iLR expressing CLL cells in vivo.
Early Clinical and Pharmacokinetic Results of Lower Dose Fludarabine and Cyclophosphamide, and High Dose Rituximab (FCR-Lite) for Patients with Untreated Chronic Lymphocytic Leukemia (CLL).
Authors: Tarhini Ahmad A. , Land S. , Meisner D. , Pietragallo L. , Sulecki M. , Lim F. , Kiefer Gauri J. , Pinkerton R. , Obsorn J. , Sukaly S. , Schaefer P.M. , Foon Kenneth A.
Published: October 2019

Introduction

Standard FCR therapy in untreated CLL patients (F-25 mg/m2 d1–3 q 4wk; C-250 mg/m2 d 1–3 q 4wk; R-500 mg/m2 d1 q 4wk for 6 cycles) was recently reported to have complete remission (CR) of 70% and overall responses (OR) of 95% (J Clin Oncol2005;23:4079). The major toxicity was grade 3/4 neutropenia during 52% of courses. One approach to decrease neutropenia without compromising efficacy could be by reducing the doses of F and C and increasing the dose of R.

Methods

We conducted a phase II study for previously untreated advanced CLL patients treated with FCR-Lite (F-20mg/m2 d1–3 q 4 wk; C-150 mg/m2 d1–3 q 4 wk; R-500mg/m2 d1 and d14 q 4wks; maintenance R-500 mg/m2 q 3 months until progression). A Simon two-stage design was used where 15 patients were accrued in the first stage and because of acceptable toxicity and response rate in stage I an additional 35 patients will be treated. The primary endpoint was response rate.

Results

Twenty eight patients (19 male, 9 female), age 36–85 years (median 58) were treated with a total of 154 FCR-Lite courses. All 28 patients were evaluable for toxicity. Grade 3/4 neutropenia occurred during 12 (8%) courses. There was one episode of neutropenic fever. Grade 3/4 thrombocytopenia occurred during 4 (3%) courses. Grade III anemia occurred during 2 (1%) courses. One patient had pneumonia. Among the 21 evaluable patients for response, the CR rate was 86%, PR rate was 14% with an OR rate of 100%. Seventeen of the CR patients were tested by flow cytometry and had no evidence of CD5+/CD19+ cells in their bone marrow after therapy. Preliminary data from pharmacokinetics performed on 16 patients showed time dependent increase in trough and peak antibody concentrations throughout treatment (Table 1).

Conclusions

Our results in the first 28 patients suggest FCR-Lite is highly effective with considerably less grade 3/4 neutropenia than standard FCR. Complete responders had no detectable CD5+/CD19+ cells in their bone marrow following FCR-Lite.

Table 1. Serum Rituximab Concentrations Before and After Infusion (N=16 patients) Infusion No. Concentration (μg/ml) (Mean) C1,d1, before 0 C1,d1, after 116.1 C1,d14, before 10.0 C1,d14, after 207.8 C2,d1, before 33.2 C2,d1, after 231.4 C3,d14, before 125.3 C3,d14, after 345.5 C4,d1, before 173.2 C4,d1, after 390.8
Oligonucleotide Microarray Analysis of Gene Expression Profiles in Chronic Graft-Versus-Host Disease (GVHD) after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Oh Sukjoong , Lee Young Ju , Mee Kwon Soo , Park Eunkyung , Cho Sungbum , Kim In Ho , Yoon Sung Soo , Kim Joo Han , Park Seonyang
Published: October 2019

Chronic graft-versus-host disease (GVHD) is one of the most serious long-term complications following allogeneic hematopoietic stem cell transplantation. The pathophysiology of chronic GVHD is poorly understood. Certain genes may affect the outcome by modulating tissue injury with the alloimmune reaction. We assessed the gene profiles of 21 transplant recipients with oligonucleotide microarray (CodeLink®) containing 20,142 probes. All patients received hematopoietic stem cells from HLA-matched sibling donors except one who’s HLA was mismatched in one A locus. All patients were in complete remission and in complete chimerism state. Among the 21 patients, 11 had chronic GVHD and 10 not. RNAs were extracted from peripheral blood mononuclear cells of the transplant recipients. Sample-wise hierarchical clustering of the whole gene expression profile showed tendency of GVHD patients aggregating in the dendrogram. In GVHD patients, 141 genes were revealed up-regulated by SAM method. Gene ontology annotation of these genes indicated that up-regulated genes were related with proteolysis, lipid biosynthesis, phosphate metabolism and tissue development. A total of 461 pathways were examined to find the pathways showing differential expression patterns between GVHD and non-GVHD patients. The results showed that 227 pathways were statistically significant. These pathways included many immune-related processes, including T cell receptor signaling pathway, T helper cell surface molecules pathway, and HIV induced T cell apoptosis. With the 17 selected classifier genes, prediction accuracy of PAM algorithm was 86%. We could identify differentially expressed genes and pathways in chronic GVHD patients using oligonucleotide microarrays. We also found that extracted classifier genes showed relatively high prediction accuracy. Gene expression profile data may provide new insights into biological mechanism underlying GVHD and reveal disease-associated biomarkers of potential therapeutic targets.
Differences in Immune Reconstitution and Cytokine Production in Children with Sibling Donor BMT Versus T-Cell Depleted PBSCT from Unrelated or Mismatched Family Donors.
Authors: Wiegering Verena A. , Eyrich Matthias , Schlegel Paul G. , Winkler Beate
Published: October 2019

Introduction: Transplantation of haematopoietic stem cell (HSCT) from human leucocyte antigen (HLA)-disparate parental donors presents an approach for the treatment of patients lacking an HLA-matched donor, but little is known about differences in immune reconstitution as compared to conventional BMT. We prospectively compared paediatric recipients of positively selected CD34+ peripheral blood stem cells from unrelated or HLA-mismatched donors vs. recipients of unmanipulated bone marrow from matched sibling donors.

Patients: Immune reconstitution was studied in 20 children (20 transplants [5 sibling, vs. 8 UD, 7 haplo]; median age was 9,6 years; range 7m-26y; 7 female, 13 male). Blood samples were drawn before allo-HSCT, on days 14 (take), 30, 60, 100, 200 and 365 and >15months after SCT.

Methods: We analyzed lymphocyte subpopulations using flow cytometry. Cytokines (IFNg, IL2, TNFa, IL4, IL5, IL10) were determined by FACS after in vitro stimulation with PMA, ionomycin and brefeldin for 24h. Additionally, we measured IL2, IL7, IL13, IL15, IFNg and TGFb in unstimulated sera by ELISA. Further more, we studied TREC values and did spectratype analysis by real time-PCR and gel electrophoresis.

Results: After allo-HSCT, NK-cells were the cells, which regenerated first. In T-cells, decreased absolute counts could be detected as well as an inverted CD4-CD8-ratio during the first year after SCT. In CD4+ T-cells, the memory phenotype (CD45RO+) predominated. Sibling recipients show a faster T-cell regeneration than recipients of UD or mismatched family donors (MM).

As to cytokine levels in unstimulated sera, IFNg levels remained stable, while we saw high level of IL4 shortly after allo-HSCT; IL4 levels decreased during the first year after SCT. TGFb showed increased levels post-transplantation for up to two years. In stimulated T-cells, we measured a rise in Th2-cytokines (IL4, IL5, IL10) until d60 and a decrease of IFNg/TNFa. Th2-cytokines returned to pre-transplantation levels until d200, whereas Th1-cytokines rise to higher level than before transplantation. We could find that IL2 was produced predominantly by CD4+ / IFNg+ cells and by CD8+cells. Patients who received T-cell depleted stem cell preparations show lower IFNg and TNFa values and higher Th2-cytokine levels than patients who received bone marrow.

TREC counts appear earlier in sib HSCT (median d60) than in MM-transplant (median d200). As to spectratype values, we found a distribution of the 24Vb gene families of 4,18% per Vb-family with clonal expansion, especially in the first months after HSCT, of BV6b>BV23>BV1>BV16.
Clinical and Pathological Differences between Nasal and Nasal-Type NK/T Cell Lymphomas: A Summary of 136 Cases from the International T Cell Lymphoma (ITCL) Project.
Authors: Au Wing , Intragumtornchai Tanin , Nakamura Shigeo , Armitage James Olen , Liang Raymond
Published: October 2019

Background: Due to their lower incidence, pathological heterogeneity and varied geographical distribution, the clinicopathological features and prognostic factors in mature T/NK cell lymphomas have not been comprehensively studied in international studies.

Materials and methods: Consecutive adult new cases of mature T cell neoplasms from 1990–2002 from 21 clinical centers in 13 countries were reviewed. NK/T nasal/nasal type lymphoma was defined by standard WHO criteria after central review.

Results: Among 1159 cases of proven T cell lymphomas, 136 cases (11.7%) of NK/T lymphomas were registered (nasal 68%, nasal-type 26%, aggressive or unclassified 6%). Its incidence among T cell lymphoma was higher in Oriental (22%) than in Western countries (4%) and in Continental Asia (43%) than in Japan (11%, 19% excluding ATLL), where nasal-type subcategory is particularly uncommon (6% vs 42%). The median age was 49 years with male to female ratio of 2:1. The nasal-type cases had higher stage (p=0.0002) and LDH (p=0.01), more B symptoms (p=0.042), bulky disease (p=0.026) and poorer performance status (p<0.001) than nasal ones. Compared to nasal-type cases, more nasal patients received treatment (98% vs 80%, p=0.0009), including anthracycline (89% vs 76%, p=0.08) and radiotherapy (RT, 52% vs 24% p=0.018). The inclusion of RT to early stage nasal cases yielded survival benefit (p=0.045). The median overall survival (OS) was inferior in nasal-type than nasal cases for both early (0.36 vs 2.96 yr, p<0.001) and late stage diseases (0.28 vs 0.8 yrs, p=0.031). The event free and overall survival curves tend to overlap, signifying poor salvage efficacy for relapses. Among nasal disease, prognostic value for OS was confirmed for all three published indices: IPI (Schipp et al, p=0.0057), T-IPI (Went et al, p=0.044) and NK/T PI (Lee et al, p<0.001). In addition, Ki67 staining >50% (p=0.05), transformed T cell >40% (p=0.022), raised CRP (p=0.025), hemoglobin <11g/dl (p=0.0038), platelet count <150×109/l (p=0.0092), B symptoms (p=0.042) were associated with poorer OS on univariate analysis. Their prognostic values were retained on multivariate analysis, after controlling for factors used for the IPI, T-IPI and NK/T PI. However, none of the histological or clinical prognostic factors or prognostic indices was significant for nasal-type disease.

Conclusion: The prognosis of nasal/nasal-type lymphoma is distinctly worse than other categories of peripheral T cell lymphomas. The clinical feature, treatment response and epidemiology of nasal type lymphoma is different from that of nasal NK lymphoma and the poor response preclude the finding of any favorable prognostic factors. There are still grounds for developing a better prognostic model for nasal cases to triage patients to more aggressive or novel treatment.
Treatment of Adenovirus Infection by Adoptive Immunotherapy: Adenovirus Capsid Hexon Is the Main Target Protein of Adenovirus-Specific Cellular Immunity.
Authors: Arbach Olga , Frentsch Marco , Chmielewicz Barbara , Kaiser Marco , Ellerbrock Heinz , Lauster Roland , Arnold Renate , Radbruch Andreas , Voigt Sebastian , Ebell Wolfram , Thiel Andreas
Published: October 2019

Introduction: Adenovirus (AdV) infection is a severe complication after hematopoietic stem cell transplantation, particularly in paediatric patients. Control of AdV infection particularly seems to be dependent on CD4+ T-cells but their main AdV antigenic targets have remained unknown so far. In order to design protocols for targeted adoptive immunotherapy the identification of the main AdV antigenic targets is a fundamental prerequisite.

Methods: We here adapted a novel technique to directly assess the entire repertoire of CD4+ T-cells specific AdV antigens according to antigen-induced CD154 expression after short-term ex vivo stimulation. AdV-lysate-specific and AdV-hexon-specific CD4+ T-cells were isolated, expanded in vitro and further assessed for their fine specificities using recombinant AdV-proteins and AdV-lysates from various AdV-serogroups. The cytokine profile of AdV-lysate- and AdV-hexon-specific CD4+ T-cells were assessed by intracellular analysis of AdV-induced CD154+ CD4+ T-cells.

Results: AdV-lysate-specific CD4+ T-cells reacted predominantly with AdV-hexon capsid protein. Furthermore, AdV-hexon (serogroup B) specific CD4+ T-cells crossreacted with recombinant hexon protein derived from various other AdV-serotypes and were characterized by a Th1-like cytokine profile.

Conclusion: Our results prove the effectiveness of antigen-induced CD154-expression for assessment of the entire repertoire of CD4+ T-cells specific for pathogens, for the identification of immunodominant target antigen from pathogens. We demonstrate that adenovirus hexon protein is a suitable candidate antigen for the ex vivo generation of adenovirus-specific, serogroup cross-reactive CD4+ T-cells with a Th1-like cytokine profile for adoptive T-cell therapies.
Randomized Comparison of 4-Times-Daily Versus Once-Daily Intravenous Busulfan in a Conditioning Therapy for Hematopoietic Cell Transplantation (HCT).
Authors: Ryu Seong-Gil , Lee Je-Hwan , Choi Seong-Jun , Lee Jung-Hee , Lee Young-Shin , Seol Miee , Hur Eun-Hye , Lee Soo Han , Bae Kyun Seop , Noh Gyu Jeong , Lee Moo-Song , Yun SungCheol , Han Sang Beom , Lee Kyoo Hyung
Published: October 2019

Several single-arm studies have shown that once-daily or twice-daily intravenous busulfan (iBU) may be equally safe and effective compared to traditional 4-times-daily iBU. We performed a prospective randomized trial of 4-times-daily vs. once-daily dosing of iBU in a conditioning therapy for HCT, and compared pharmacokinetic (PK) characteristics of iBU and clinical outcomes in terms of engraftment, post-HCT toxicities, non-relapse mortality (NRM), and overall survival. All of 60 patients, who received iBU on the first day of conditioning therapy for HCT, were randomized to receive iBU either as 0.8 mg/kg over 2 hr 4 times a day (BU4 arm) or 3.2 mg/kg over 3 hr once a day (BU1 arm) between May 2004 and August 2005. Limited sampling strategy was used for PK studies, which were done for the first busulfan dosing. Baseline patient and donor characteristics were well balanced between 2 arms regarding sex, age, diagnosis of underlying disease, time from diagnosis to HCT, disease status at HCT, GVHD prophylaxis regimen, conditioning therapy regimen, ABO mismatch between donor and recipient, donor-recipient sex pair, donor age, type of graft, donor type, and cell doses of graft. The complete PK data were obtained from all 60 patients and those were comparable between 2 arms: elimination half-life (mean ± SD) was 2.75 ± 0.22 vs. 2.83 ± 0.21 hr in BU4 arm vs. BU1 arm, respectively (P=0.304), estimated AUC was 7933.43 ± 2177.71 vs. 8556.86 ± 2452.07 μM × min per day (P=0.367), and clearance was 2.05 ± 0.36 vs. 1.91 ± 0.31 ml/min/kg (P=0.133). Median times to engraftment of neutrophils (BU4 arm vs. BU1 arm, 14 vs. 14 days, P=872) and platelets (26.5 vs. 25.5 days, P=0.946) after HCT were similar between 2 arms. There were no significant differences in cumulative incidence of acute GVHD (BU4 arm vs. BU1 arm, 31.0% vs. 13.8%, P=0.145) and occurrence of CMV infection (40.0% vs. 23.3%, P=0.165) or hepatic veno-occlusive disease (10.0% vs. 16.7%, P=0.448). Other toxicities within 100 days after HCT were not significantly different between two arms. Cumulative incidence of NRM was 25.5% vs. 17.3% (P=0.488) and overall survival probability at 1 year was 69.0% vs. 70.0% (P=0.758). In conclusion, our randomized study demonstrates that once-daily iBU has similar PK profiles and results in at least not inferior clinical outcomes compared to traditional 4-times-daily iBU.
Voriconazole Therapeutic Drug Monitoring: Relationship between Drug Levels and Liver Function Tests.
Authors: Trifilio S. , Khalid M.K. , Pennick G. , Pi J. , Bainbridge N. , Zook J. , Mehta J.
Published: October 2019

Voriconazole is increasingly being used after HSCT. The hepatic cytochrome P450 isoenzyme 2C19 plays a significant role in voriconazole metabolism. As CYP2C19 exhibits significant genetic polymorphism, some patients metabolize voriconazole poorly resulting in increased plasma drug levels. However, the clinical significance of this is unknown. There is some evidence that toxicity rates are higher in patients with higher voriconazole levels (Boyd et al. Clin Infect Dis2004;39:1241–1244). In a preliminary study of 41 voriconazole levels in 25 patients, we had found that voriconazole levels correlated with aspartate aminotransferase (AST) and alkaline phosphatase (AP) levels (Trifilio et al. Bone Marrow Transplant2005;35:509–513). It was unclear if the abnormal liver function was the cause or the result of higher voriconazole levels. To further elucidate this relationship, we analyzed data on 171 steady-state plasma trough levels performed after at least 5 days of voriconazole therapy in 87 patients with hematologic malignancies. There were 1–5 levels per patient (median 2). Most patients had undergone allogeneic hematopoietic stem cell transplantation. Drug levels were monitored using HPLC (Pennick et al. Antimicrob Agents Chemother2003;47:2348–2350). Of the 201 samples assayed, 30 were below the detection limit of the assay (0.2 μg/mL), and were excluded. The daily voriconazole dose (divided into 2) was 200 mg (n=3), 400 mg (n=129), 500 mg (n=18), 600 mg (n=15), or 800 mg (n=6); corresponding to 2.0–13.3 mg/kg (median 5.3). The voriconazole levels were 0.2–12.5 μg/mL (median 1.7). The table shows the correlation between voriconazole levels, and weight, dose and biochemical parameters individually.

However, in multivariate regression analysis, the parameters found to correlate significantly with voriconazole levels were ALT (P=0.0005), AST (P=0.003), and AP (P=0.027). The relationship with albumin was of borderline significance (P=0.062). Importantly, the daily dose of voriconazole in mg or in mg/kg was not predictive of drug levels. This larger data set confirms our previous observation that there is a significant relationship between elevated liver function tests and higher voriconazole levels. However, because of the relatively high frequency of abnormal liver function tests in such groups of patients, the cause-effect relationship still remains uncertain. These data suggest that pending further clarification, voriconazole levels may need to be monitored in patients with significantly abnormal liver function tests.

Parameter Median (range) r P Dose (mg) 400 (200–800) 0.19 0.013 Weight (kg) 80 (39–135) 0.18 0.018 Dose (mg/kg) 5.3 (2.0–13.3) 0.23 0.002 ALT (IU/L) 25 (4–608) 0.10 0.25 AST (IU/L) 25 (6–524) 0.14 0.11 AP (IU/L) 95 (27–920) 0.27 0.002 Bilirubin (mg/dL) 1.1 (0.1–17.3) 0.01 0.89 Albumin (g/dL) 2.4 (0.8–3.9) 0.28 0.001 Creatinine (mg/dL) 1.1 (0.2–10.1) 0.01 0.92
Influence of Killer Immunoglobulin-Like Receptor (KIR) Matching on Achieving T Cell (CD3+) Complete Donor Chimerism (CDC) in Related Donor Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplantation (NMHSCT).
Authors: Sobecks Ronald , Ball Edward J. , Askar Medhat , Theil Karl , Rybicki Lisa , Thomas Dawn , Bates Jennifer , Kalaycio Matt , Andresen Steven , Pohlman Brad , Dean Robert M. , Sweetenham John William , Bernhard Laura , Cherni Kelly , Maciejewski Jaroslaw P. , Bolwell Brian J.
Published: October 2019

The reactivity of NK cells and some T cell populations is regulated by KIR interactions with HLA class I molecules. Such interactions have been suggested to influence outcomes after myeloablative allogeneic HSCT. However, in NMHSCT the effect of KIR interactions on outcomes including the development of CDC has not been well described. We analyzed 51 pts who received related donor NMHSCT at our institution from 1/10/00–10/25/05. All pts received fludarabine 30 mg/m2/d x 3 days followed by total body irradiation 200 cGy (n=35) or 400 cGy (n=16) for conditioning. The median age was 54 (range, 21–64). Short tandem repeat analysis for T cell (CD3+) chimerism was performed on peripheral blood and CDC was defined as achievement of >95% DNA of donor origin in the CD3+ T cells. 37 (73%) of patients achieved CDC at a median time of 3.3 mos (range, 0.4–11.2). KIR genotypes were determined for recipients by PCR-rSSOP analysis. Donor HLA KIR ligands were categorized as:

HLA-Cw groups C1 (+ or −); C2 (+ or −); HLA-Bw4 (+ or −); and HLA-A3 or -A11 (+ or −) [as reviewed by Farag et al. Blood2002; 100:1935–47].

Recipient KIR genotype and donor HLA KIR ligands were used to generate an inhibitory KIR score for pts from 1 to 4 corresponding to the potential number of inhibitory KIRs engaged. 7 pts had a score of 1, 27 had a score of 2, 14 had a score of 3 and 3 had a score of 4.

Figure Figure

The Kaplan-Meier method was used to estimate the achievement of CDC by inhibitory KIR score (figure, p=0.09). Pts with a score of 1 were less likely to achieve CDC compared to those with a score of 2 (p=0.02), while those with a score of 2 tended to be less likely to develop CDC than those with a score of 4 (p=0.07). There were no differences in CD34+ or CD3+ cell doses between any of the groups. When combined with the inhibitory KIR score data the presence of single or multiple activating KIR’s was not found to influence the development of CDC. Thus, pts with lower inhibitory KIR scores may have more active anti-donor effector cells (NK cells and T cell subsets) that may reduce donor cell chimerism. Conversely, those with higher inhibitory KIR scores may have less active populations and be more likely to achieve CDC. Given the genotypic potential to inhibit all NK cells KIR expression may be variable among different clones, and may affect the development of CDC. Further investigation of KIR expression at the cellular level rather than by genotyping alone should be pursued.
Severe Alterations of Bone Mass and Bone Structure after Allogeneic Stem Cell Transplantation: An Osteopathologic Analysis.
Authors: Massenkeil Gero , Fiene Cornelia , Rosen Oliver , Doerken Bernd , Arnold Renate , Delling Guenther
Published: October 2019

Prolonged survival after allogeneic SCT has led to an increased awareness of late complications in these patients. Loss of bone density after SCT has been reported in radiologic analyses (Ebeling 1999, Schulte 2000, Massenkeil 2001). We have studied bone mass and structure alterations in bone marrow biopsies after SCT.

Patients and Methods: 67 patients after allogeneic SCT were included in this analysis. Median age was 36 years (range 17–56), 35 male, 32 female. Diagnoses were: ALL 27 patients, AML 14, MDS 6, CML 20. 38 patients were in 1. CR or 1. cP, 29 were transplanted with advanced disease. 64 received standard high-dose conditioning and 3 were transplanted after reduced intensity conditioning. 42 received bone marrow and 25 mobilized peripheral blood as stem cell source.40 patients were transplanted from HLA-identical related and 27 from unrelated donors.

31 bone marrow biopsies underwent Micro-CT (Scanco, Zürich, CH) and were analysed after 3-D reconstruction. Usual parameters of trabecular structure were investigated.

Results: Median bone volume/tissue volume (BV/TV), which serves as a parameter of histopathologic bone mass was 13.7% before SCT (range 8.6–44.4%), well below 18%, which are considered the osteopathologic threshold for osteoporosis. At first follow-up 6–9 months after SCT, BV/TV had dropped to 10.1% (5.9–24.6%) and at 1–2 years after SCT BV/TV was still below the pre-SCT values at 12.2% (6.1–19.3%).

Number of trabecules (Tb.N.) dropped from 1.63/mm (range 0.93–2.32/mm) to 1.34 (1.01–5.61/mm) after 6–9 months and slightly increased to 1.54 (1.26–1.88/mm) after 1–2 years.

Trabecular thickness (Tb. Th.) fell from 0.20 mm before SCT (normal Tb. Th. 0.20–0.30 mm) (range 0.16–0.49 mm) to a minimum of 0.16 mm (0.14–0.25 mm).

As a consequence, intertrabecular space (Tb.Sp., normal appx. 0.60 mm) increased from 0.60 mm (0.36–1.11 mm) to 0.73 mm (0.23–1.04 mm) after SCT.

Discussion: Independently from underlying diagnosis trabecular parameters changed within the first 6 months after SCT, indicating early loss of bone mass and structure. Afterwards, they showed some tendency to normalization, however, none of the parameters went back to baseline values. Number of patients analysed over time was to small to find out causative clinical parameters like chronic GvHD. Radiologic bone density was available for these patients and showed synchronous variations over time.

Conclusions: Structural changes of trabecular bone develop early after allogeneic SCT and underscore the severeness of bone loss observed in these patients. Even after prolonged follow-up, structural changes and diminished bone mass persist and indicate a long-term cause of morbidity in these patients.
Myeloablative Allogeneic Stem Cell Transplantation for Relapsed Composite Low and Intermediate Grade Lymphoma Is Not Superior to Autologous Stem Cell Transplantation - Reduced Relapse Risk Is Offset by Higher Treatment Related Mortality.
Authors: Ramadan Khaled M. , Connors Joseph M. , Al-Tourah Abdulwahab J. , Gascoyne Randy D. , Song Kevin , Barnett Michael J. , Nantel Stephen H. , Nevill Thomas J. , Shepherd John D. , Sutherland Heather J. , Lavoie Julye , Forrest Donna L. , Hogge Donna E. , Voss Nicholas J. , Smith Clayton A. , Toze Cynthia L.
Published: October 2019

No reports compare autologous and allogeneic stem cell transplantation (auto-SCT vs allo-SCT) for patients (pts) with relapsed (REL) composite low and intermediate grade non-Hodgkin lymphoma (L/I-NHL). Fifty four pts with composite L/I-NHL (30 transformed, 16 composite(same site), 8 discordant(different sites)) who underwent allo (n=40) or auto-SCT (n=14) from Jan ‘89 to June ‘05 were evaluated (Table 1) to compare long-term results.

Twenty pts (37%) (11/40 allo and 9/14 auto) are alive with median follow-up of 32 months (range 10–87). The 2 and 5-y OS for the whole group were 43% [95% confidence interval: 28–56%] and 29% [14–46%] respectively; the 2 and 5-y EFS 40% [26–53%] and 24% [11–39 %]. 5-y OS for the auto and allo-SCT were 51% and 23% respectively (p=.09)(Fig. 1). The 2 and 5-y EFS for the allo vs auto-SCT groups were 36% [24–55%] vs 54% [33–89%] and 27% vs 23% (p=0.4).

Thirty four pts (63%) died; 18 (33%) with REL NHL and 16 (30%) with TRM. TRM was due to GVHD (n=7), infection (n=6), cerebral edema (n=1) and veno-occlusive disease (n=1). All but one (cerebral edema) TRM deaths occurred post allo-SCT. The cumulative incidence (CI) of TRM and REL for the whole group were 37% [17–57%] and 47% [30–64%]. The CI of TRM for allo and auto-SCT were 47% [20–74] and 8% [0–23%] respectively (p=.009)(Fig. 2).

Twenty pts (37%) relapsed post SCT (14/40, 35% allo and 6/14, 43% auto). CI of REL NHL for allo and auto-SCT were 42% [23–61%] and 65% (p=.008)(Fig. 2).

In conclusion EFS for composite L/I-NHL is low post auto-SCT due to a high relapse rate despite acceptable TRM. While relapse risk is significantly lower post allo-SCT, this advantage is offset by a higher TRM in these pts. Future attempts to reduce TRM while preserving the allogeneic graft vs lymphoma effect may be beneficial.

Table 1. The clinicopathological characteristics of 54 patients treated with auto and allo-SCT for relapsed composite L/I-NHL Parameter Allo-SCT n=40(%) Auto-SCT n=14(%) * at diagnosis Age: Range (Median) 28–57(44) 25–59(49) M:F 2:1 1.3:1 Diagnosis Transformed lymph. 25(63) 5(36) Discordant lymph. 7(17) 1(7) Composite lymph. 8(20) 8(57) Initial Stage I/II 4(10) 6(42) III/IV 36(90) 8(58) B symptoms* 11(28) 6(43) BM involvement* 29(73) 3(21) IPI* 0–1 18(45) 11(79) 2–3 21(53) 3(21) 4–5 1(2) 0 Prior treatment CHOP 33(83) 14(100) Purine analogue 17(43) 2(14) Rituximab 12(30) 6(43) Fig 1: OS for auto/alle groups Fig 1:. OS for auto/alle groups Figure Figure
Clinical Efficacy of Lenalidomide {Revlimid®} in Patients with Relapsed or Refractory Chronic Lymphocytic Leukemia (CLL): Updated Results of a Phase II Clinical Trial.
Authors: Chanan-Khan Asher Alban , Miller Kena C. , Musial Laurie , Padmanabhan Swaminatha , Lawrence David , Bernstein Zale P. , Takeshita Kenichi , Spaner David , Byrne Catriona , Chrystal Cynthia , Czuczman Myron S.
Published: October 2019

Introduction: Lenalidomide (L) is an immunomodulatory agent (IMiD®), recently approved for treatment of patients (pts) with previously treated multiple myeloma and del 5q- MDS. Its antitumor effects are reported to be mediated at least in part through modulation of both the cytokine and the immune cellular tumor microenvironment. Clinical efficacy of L in pts with B-CLL has not been investigated previously. In a phase II clinical study, we examined the antileukemic effects of L in pts with relapse (rel) or refractory (ref) B-CLL.

Patients and Methods: CLL pts with rel or ref disease were eligible. L was given at 25mg PO QD from days 1–21 of 28-day cycle. Response was assessed every month using the NCI-WG 1996 criteria. Treatment with L was continued until progressive disease (PD) or molecular complete response (CR). Pts with PD received rituximab (R) (375mg/m2) with L. Target enrollment was 45 patients.

Results: Forty-five pts, median age of 64 years (range: 42–75) were enrolled. Advance stage disease was noted in 64%, elevated B2M in 24% and fludarabine refractory disease in 51% of the pts. Pts had a median of 3 prior therapies (range 1–10). All pts are evaluable for toxicity. Thrombocytopenia and neutropenia were the most common hematologic toxicity while fatigue and flare reaction (sudden, tender enlargement of lymph node, liver and/or spleen, with or without rash, low grade fever and/or increase in white blood counts) were the most common non-hematologic complications. Two patients experience reversible tumor lysis syndrome. On an intent-to-treat analysis, overall response rate was 42% with complete remission in 9% (n=4) and partial remission in 33% (n=15) of the pts. Molecular complete response (determined by PCR) was noted in 3 pts. Twelve pts were inevaluable (unable to complete 2 treatment cycles; either for toxicity or consent withdrawal) 4 are too early for response evaluation. Response rate among evaluable patients was 65.5% (19/29). To date only 2 pts have required the addition of R to L due to PD. Both have achieved a PR with combination therapy.

Correlative studies: we evaluated baseline T and NK cell repertoire, modulation of cytokines (VEGF, IL-10, IL-6, TNF-alpha) as well as modulation of gene expression profiles before and after treatment with L, this data will be presented at the meeting.

Conclusion: L is clinically active in pts with rel/ref CLL. CR’s, as well as, molecular CR determined by PCR were achieved with single agent L. Interestingly, in pts who developed resistance to L alone, addition of R resulted in a clinical response. These results are exciting and warrant further investigation of L in CLL pts. Final results of this study will be presented at the meeting.
Rapid and Wide Immunereconstitution Obtained with HSV-TK Engineered Donor Lymphocyte Add-Backs Permits Long-Term Survival after haplo-HSCT.
Authors: Bonini Chiara , Ciceri Fabio , Stanghellini Maria Teresa Lupo , Bondanza Attilio , Magnani Zulma , Perna Serena Kimi , Bernardi Massimo , Peccatori Jacopo , Servida Paolo , Crippa Fulvio , Kaneko Shin , Valtolina Veronica , Salomoni Monica , Turchetto Lucia , Toma Salvatore , Traversari Catia , Bruzzi Paolo , Castagna Luca , Santoro Armando , Apperley Jane , Slavin Shimon , Colombi Scialini , Stampino Corrado Gallo , Bregni Marco , Bordignon Claudio
Published: October 2019

T-cell depletion of allogeneic hematopoietic stem cell graft (HSCT) represents the most powerful approach to prevent graft-versus-host disease (GvHD), thus allowing to overcome HLA barriers in patients with high risk malignancies, lacking a conventional donor. We hypothesized that early add-back of suicide-gene transduced donor lymphocytes (TK cells) to leukemic patients undergoing haploidentical HSCT (haplo-HSCT) could provide early immune-reconstitution and selective control of GvHD. In a phase II clinical trial (protocol MMTK007), 17 of 29 enrolled pts, (median age 52), received add-backs of 10^7/kg TK cells 42 days after haplo-HSCT. TK cells engraftment, observed in 14 patients, was necessary and sufficient for a rapid and effective immunereconstitution (IR), with a median of 144 (101–336) CD3+, 59 (28–149) CD4+ and 86 (52–279) CD8+ cells/mcl at day 100 after HSCT. Accordingly, engraftment of TK cells was tightly correlated with clinical outcome: while 3/3 pts who failed TK cells engraftment died of infections, only 1/14 TK engrafted patients died from infections (last event at day +166). As shown in Table I, the immune repertoire of treated patients improved significantly at 6 months post transplant and normalized completely in 12 months.

High numbers of T cell precursors specific for CMV and EBV were detected at immune reconstitution (median of 86 and 69 gIFN specific spots/10^5 PBMC respectively) and predicted subsequent freedom from viral reactivation (p=0.002). Six pts developed acute (GvHD), (grade I to IV) that was always completely abrogated by the suicide system. Overall survival of TK cells treated patients is 50% at three years.

These results indicate that TK-DLI abolish late mortality after CD34+ haplo-SCT in adults. A phase III multicentric study will start in 2007 to validate prospectively the advantage of TK-DLI in haplo-SCT.
Sequential Heart and Autologous Stem Cell Transplantation for AL Amyloidosis.
Authors: Lacy Martha , Dispenzieri Angela , Hayman Suzanne R. , Kumar Shaji , Kyle Robert , Rajkumar S. Vincent , Edwards Brooks S. , Rodeheffer Richard J. , Robert Frantz P. , Kushwaha Sudhir S. , Clavell Alfedo L. , Dearani Joseph A. , Sundt Thoralf M. , Daly Richard C. , McGregor Christopher G. A. , Gastineau Dennis A. , Gertz Morie Abraham
Published: October 2019

Background: Patients with AL and congestive heart failure have a median survival of less than one year.

Methods: Between 1994 and 2005, 11 patients underwent sequential heart transplant followed by autologous peripheral blood stem cell transplant (SCT) for treatment of AL. Patients had heart dominant AL with minimal/no other organ impairment and no evidence of multiple myeloma.

Results: Patients ranged in age from 35 to 63 years (median 54). All had biopsy proven amyloidosis and monoclonal protein detectable in serum (73%) or urine (100%). Free light chain assay was available prior to SCT in 6 and was abnormal in all. All patients presented with NYHA Class III or IV heart failure. All but two patients had no other organ involved at the time of heart transplant. One patient had his heart transplant at another facility and had renal involvement at the time of heart transplant. One patient had peripheral nerve involvement documented prior to heart transplant. Five patients had progression of amyloid to involve other organs by the time of SCT. These included: renal (6), peripheral nerve (2), macroglossia (3), gastrointestinal (2). The median time from diagnosis to heart transplant was 3 months (range 1 to 5). The median time from heart transplant to SCT was 8 months, (range 3 to 24). Conditioning chemotherapy consisted of melphalan 200 mg /m2 (6 patients) or melphalan 140 mg/m2 (5 patients). Two patients died of complications before day +100, (TRM of 18%). Hematologic remissions were seen in 8 and organ responses in 3. Five were unable to be evaluated for organ response due to the heart transplant. Three died from progressive amyloidosis at 66, 56.7, and 55 months following SCT. The median survival is 56.7 months from SCT. Six are alive with a median follow-up of 24 months, range 9 to 90 months. Among the 6 surviving patients, one patient relapsed and one patient has failed to improve, but 4 patients are in continued remission. One patient developed secondary myelodysplastic syndrome.

Conclusions: Heart transplant followed by SCT is feasible and offers durable remissions for carefully selected patients with cardiac AL.

Figure Figure