bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoantibodies Specific to hnRNP K: A New Diagnostic Marker for Immune Pathophysiology in Bone Marrow Failure Syndromes.
Authors: Qi Zhirong , Takamatsu Hiroyuki , Lu Xuzhang , Espinoza J. Luis , Sugimori Naomi , Mochizuki Kanako , Nakao Shinji
Published: April 2020

The pathophysiology of bone marrow failure syndromes (BMS) such as aplastic anemia (AA) and refractory anemia (RA) of myelodysplastic syndromes defined by the FAB classification involves immune mediated bone marrow injury and intrinsic defects of hematopoietic stem cells. The diagnosis of the immune pathophysiology is essential in providing the optimal treatment to patients with BMS. In order to identify a new diagnostic marker for the immune pathophysiology of BMS, the sera of AA patients characterized by the presence of small populations of paroxysmal nocturnal hemoglobinuria-type (PNH) cells were screened for the presence of antibodies (Abs) recognizing proteins derived from a megakaryocytic leukemia cell line, UT-7. Two-dimensional electrophoresis of UT-7 lysates followed by immunoblotting revealed a distinct spot of 65 kDa. Peptide mass fingerprinting identified this protein as a heterogeneous nuclear ribonucleoprotein (hnRNP) K. Although hnRNP K is known to be ubiquitously expressed, the expression levels based on a Western blotting analysis were greater in several leukemia cell lines such as UT-7, K562 and OUN-1 in comparison to peripheral blood mononuclear cells. When the sera of newly diagnosed 87 BMS patients (62 with AA and 25 with RA) were examined for the presence of anti-hnRNP K Abs using an enzyme-linked immunosorbent assay (ELISA) with recombinant hnRNP K protein, significantly higher titers of anti-hnRNP K Abs were detected in 13 (34%, 10 with AA and 3 with RA) of 38 patients with BMS displaying increased PNH-type cells (PNH+) and in 15 (31%, 8 with AA and 7 with RA) of 49 patients without increased PNH-type cells. The presence of hnRNP K Abs was significantly correlated with the presence of anti-diazepam-binding inhibitor-related protein-1 (DRS-1) Abs (r=0.7206) and anti-moesin Abs (r=0.8318). Of the 87 patients, 49 patients (39 with AA and 10 with RA) received antithymocyte globulin plus cyclosporine therapy after the Ab screening. All of the 22 patients with high anti-hnRNP K Ab titers responded to therapy while 10 (37%) of 27 patients without the high titer Abs responded (P < 0.005). When the three Ab titers were assessed, the response rate to immunosuppressive therapy in patients showing high titers in at least one Ab was 96%, while the response rate in patients not showing high titers in any of the auto-Abs was 39% (P < 0.005). These findings suggest that in patients with immune mediated BMS, a breakdown of immune tolerance to multiple self antigens including hnRNP K may take place and that the detection of these auto-Abs may help diagnose the immune pathophysiology of BMS.
Depletion of the Shwachman-Diamond Syndrome Protein in Hematopoietic Progenitor Cells Impairs Growth, Colony Formation, and Ribosome Function.
Authors: Sezgin Gulay , Nihrane Abdallah , Ellis Steven , Liu Johnson M.
Published: April 2020

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, skeletal abnormalities, and bone marrow failure, which can evolve to leukemia. Mutations in SBDS have been shown to cause SDS. We and other investigators have suggested that SBDS orthologs in yeast play a role in biogenesis and function of the 60S ribosomal subunit. To clarify the unknown function of SBDS in hematopoiesis, human erythroleukemia TF-1 cells were transduced with lentiviral vectors expressing the green fluorescent protein (GFP), neomycin phosphotransferase, and small interfering RNA (siRNA) against SBDS. After transduction, cells were selected for neomycin resistance and then sorted by flow cytometry. To probe for SBDS, an antibody against the carboxyl-terminus of human SBDS was generated, and individual TF-1 cell clones expressing different siRNAs were confirmed to knock down SBDS expression by Western blot analysis. Our experiments were aimed at analyzing the cellular effects of SBDS knockdown. The growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to cells stably transduced with siRNA against a scrambled SBDS sequence. Using propidium iodide staining and flow cytometric analysis, we found an increased percentage of knockdown cells retained at the G0/G1 cell cycle phase. To address whether TF-1 cells expressing siRNA against SBDS have a selective deficiency of 60S ribosomal subunits, cell extracts were prepared and polysome profiles examined after sucrose gradient centrifugation. In preliminary experiments, TF-1 cells expressing siRNA against SBDS appeared to show a reduction in free 60S subunits and 80S subunits with a shift toward smaller polysomes, compared to cells expressing the scrambled sequence siRNA. We conclude that depletion of SBDS results in a significant growth and clonogenic defect in TF-1 hematopoietic cells. Our preliminary results also suggest defects in ribosome function and cell cycle transit, which may provide an integrated molecular explanation for the hematopoietic defect in SDS since nucleolar stress has been linked to cell cycle arrest and p53 stabilization.
Genetic Abnormalities Involved in the Development and Progression of Follicular Lymphoma.
Authors: Deffenbacher Karen E , Wright George , Iqbal Javeed , Geng Huimin , O’Shea Derville , Lister T. Andrew , Fitzgibbon Jude , Fu Kai , Liu Zhongfeng , Weisenburger Dennis , Greiner Timothy C. , Gascoyne Randy D. , Rosenwald Andreas , Campo Elias , Rimsza Lisa M. , Delabie Jan , Jaffe Elaine S , Staudt Louis M. , Chan Wing C.
Published: April 2020

Background: Follicular lymphoma (FL) is the most common indolent B-cell lymphoma and remains incurable by current therapeutic approaches. Clinical course is variable, and transformation into an aggressive lymphoma (t-FL) with marked worsening of prognosis occurs in 20–60% of patients. While Bcl2 gene translocation is a critical initiating event in the majority of FL cases, evidence indicates it is not sufficient for the development of a FL. Characterization of the genetic alterations subsequent to Bcl2 translocation will lend insight into the oncogenic pathways that contribute to FL pathogenesis and the molecular mechanisms underlying variability in clinical course.

Methods: To define recurrent genomic copy number alterations (CNA) in FL, we performed high resolution array comparative genomic hybridization (aCGH) using the Affymetrix 500K SNP array platform. aCGH data were generated on a series of 112 FL cases with available gene expression profiling (GEP) and clinical information. Gene expression data were correlated with copy number data using the Gene Expression and Dosage Integrator (GEDI) algorithm developed at the NCI.

Results: Selecting for abnormalities occurring in >10% of cases, the minimal common region (MCR) for 38 losses and 31 gains were defined. Novel common regions included gains on 15q11, 16p11, 5p14 and 19q13, and losses on 3q29, and 16p13. The MCR identified by aCGH were also compared with our existing cytogenetic data on 360 FL cases. MCR residing within the most frequent cytogenetic imbalances (>5%) were selected for analysis at the gene level to further refine these regions. These include gains on 1q21, 2p16, 7q11, 8q24, 12q13, 17q21, 18q21, 21q11, and X, and losses on 1p36, 6q, 10q, 13q34, and 17p13. Recurrent amplifications were detected for the 2p16, 15q11, and 17q21 MCR, while frequent uniparental disomy (UPD) was found to overlap the region of loss on 1p36. Recurrent UPD was also noted on 6p, 12q, 15q and 16p. For the majority of selected MCR, global expression of the genes residing in the MCR demonstrated an association with copy number status. Within these abnormalities, individual genes showing significant correlation with copy number were also identified.

Conclusion: The combination of high resolution aCGH and GEP facilitated the identification of functionally relevant genes within the chromosomal abnormalities in FL. Delineation of these molecular targets will provide insight into the oncogenic pathways that contribute to FL disease pathogenesis and may provide novel therapeutic targets.
Inversely Association of the Aiolos Transcription Factor with Clinical Progression in Chronic Lymphocytic Leukemia.
Authors: Nückel Holger , Frey Ulrich H , Sellmann Ludger , Collins Crista H , Duehrsen Ulrich , Siffert Winfried
Published: April 2020

Introduction: Aiolos encode a hemopoietic-specific zinc-finger transcription factor that is an important regulator of lymphocyte differentiation and plays a critical role in regulating B-cell development. RT-PCR analysis of the Aiolos gene expression revealed 16 Aiolos splicing variants, which have been named according to the exons missing from the full-length isoform. Recent data suggest that over 80% of expressed Aiolos in normal as well as in malignant B-cells is of the hAio1 type. Therefore, we investigated Aiolos mRNA expression (hAio1 type) in a large cohort with 155 patients along with the most commonly used biological markers in order to assess its role in risk prediction in B-CLL.

Methods and Results: The total amount of Aiolos transcripts in B-cells of 155 CLL patients using normal peripheral blood mononuclear cells represented a continuum ranging from 2.5- to 37-fold upregulation compared to that of normal B-cells, with a median of 20- fold upregulation. Moreover, Aiolos expression in B-CLL was significantly upregulated compared to cells of AML (113-fold; p<0.0001), ALL (14-fold; p=0.0024), CML (154- fold; p<0.0001), multiple myeloma (38-fold; p=0.0018) or NHL (16-fold; p<0.0001) suggesting that this Aiolos transcript is highly expressed specifically in B-CLL cells Patients with high Aiolos expression (according to ROC-analysis) had a significantly longer treatment-free survival (TFS) and overall survival (OS) than patients with low Aiolos expression (median TFS: 119 versus 45 months, p=0.005; median OS: 321 versus 244 months, p=0.0065). Evaluation of several disease characteristics in association with the Aiolos expression status of the patients’ B-CLL cells showed no significant differences for ZAP-70 expression (p=0.28), CD38 expression (p=0.067), IgVH status (p=0.49) and Binet stage (p=0.13) suggesting no correlation of Aiolos expression with these already established adverse prognostic factors. In multivariate analysis low Aiolos expression was an independent prognostic factor with significance for trend (hazard ratio 1.413; p=0.069). Sequential analyses in a subset of 10 CLL patients revealed that Aiolos expression was relatively stable over time in the majority of patients.

Conclusions: Here we demonstrate for the first time that the level of Aiolos expression is correlated with prognosis in B-CLL. The exact causes and consequences of this upregulation on the survival of CLL B-cells and the demonstrated better prognosis have yet to be determined. However, Aiolos may function as a tumor suppressor by controlling the cell cycle and DNA replication.
Overexpression of TOSO in CLL Is Triggered by B-Cell Receptor Signaling and Associated with Progressive Disease.
Authors: Pallasch Christian P , Schulz Alexandra , Kutsch Nadine , Schwamb Janine , Hagist Susanne , Wickenhauser Claudia , Ultsch Alfred , Kashkar Hamid , Hallek Michael , Wendtner Clemens-Martin
Published: April 2020

Resistance towards apoptotic stimuli mediated by overexpression of antiapoptotic factors or extracellular survival signals like B-cell receptor stimulation (BCR) are considered to be responsible for accumulation of malignant B cells in CLL . TOSO, also known as Fas-inhibitory molecule 3 (FAIM3), was identified as overexpressed candidate gene in CLL based on re-evaluation of publicly available microarray data sets. Based on primary CLL samples from 106 patients, TOSO expression was compared to healthy donor B cells using quantitative real-time PCR, western-blot, flow cytometry and immunohistochemistry. To reveal underlying mechanisms of TOSO overexpression, B-cell receptor (BCR) and CD40Ligand stimulation as well as bone marrow stroma cell co-incubation was performed. Apoptotic resistance was assessed by annexin V/7-AAD flow cytometry in context of CH11-Fas-agonistic antibody. TOSO was identified to exhibit elevated relative expression of 6.8 compared to healthy donor B cells using quantitative real-time PCR (p=0.004). High levels of TOSO expression in CLL correlated with high leukocyte count, advanced Binet stage, previous need for chemotherapy and unmutated IgVH status. CD38+ CLL subsets harboring proliferative activity showed significantly enhanced TOSO expression. Immunohistochemistry revealed upregulation of TOSO in lymph nodes of CLL patients. In lymph nodes derived from healthy donors TOSO was detected in single plasmocytoid cells within the germinal center and in the marginal zone. No specific staining was seen in follicular lymphomas. CLL-specific upregulation of TOSO was confirmed by RT-PCR, samples of follicular lymphomas, DLBCL, marginal zone lymphoma and Hodgkin cell lines did not reveal TOSO up-regulation. We evaluated functional mechanisms of aberrant TOSO expression in CLL cells and identified TOSO expression significantly being induced by BCR-stimulation compared to control cells (relative expression (RE) 8.25 vs. 4.86, p=0.013). In contrast, CD40L signaling significantly reduced TOSO expression (RE 2.60; p=0.007). Spontaneous apoptosis of CLL cells was significantly reduced by BCR-stimulus, in CD40Ligandstimulated CLL samples a slight sensitization towards Fas-mediated apoptosis was seen. In summary, we show that the anti-apoptotic factor TOSO is associated with progressive disease and enhanced in the proliferative CD38+ cell subset. Both association with unmutated IgVH and the specific induction of TOSO via the BCR suggest autoreactive BCR signaling as a key mediator of apoptosis resistance in CLL. Down-regulation of TOSO by CD40Ligand in the context of CD40Ligand-mediated Fas-sensitization of CLL reveal TOSO as a new anti-apoptotic factor in CLL. CLL-specific over-expression of the transmembrane protein might further offer new therapeutic strategies in CLL treatment.
Deregulation of Micrornas Results in Overexpression of Oncogenic Transcription Factors Involved in Pathogenesis of CLL.
Authors: Pallasch Christian P , Hagist Susanne , Patz Michaela , Schulz Alexandra , Debey Svenja , Eggle Daniela , Schultze Joachim L , Hallek Michael , Wendtner Clemens-Martin
Published: April 2020

MicroRNAs play a key role in the control of translation and mRNA degradation by binding to the 3′ untranslated regions of mRNAs. Immune cell development is especially dependent on miRNA regulation like miR-155 in germinal center development. Distinct miRNA-deregulation has been recently identified in CLL, however the proposed model of miR-15/16 mediated pathogenesis remains controversial. CLL cells of 50 treatment-naïve patients and peripheral B-cells of 14 healthy donors were separated by untouched depletion and processed for isolation of at least 200 ng total RNA. A new Illumina based Bead Chip was applied using 100 ng of total RNA for reliable hybridization. Target prediction of deregulated miRNAs was performed by in-silico predictions of miRNA-target gene interactions by TargetScan and PicTar. MiRNA target candidates were analyzed for differential protein expression in healthy donor B cells versus CLL cells. The applied technology was repetitively controlled and demonstrated to reveal reliable quantitative results. Comparing CLL samples with healthy donor B cells an overall decrease of miRNAs was observed in CLL samples. In total 19 miRNA were identified to be significantly lower expressed in CLL. 7 miRNAs were overexpressed in CLL. Comparing previously published data we could reliably identify upregulation of miR- 155 and downreguation of miR-181. However, the intensively discussed deregulation of miR-15 and miR-16 could not be verified. Furthermore we identified a so far not described CLL-specific miRNA-fingerprint of 26 miRNAs. Based on this fingerprint we analyzed for miRNA targets based on target prediction. A significantly focused number of transcription factors were identified by this primary screen. We could confirm predicted over-expression of oncogenic transcription factors by immunoblotting analysis. Targeting of deregulated miRNAs to 3′UTR of target genes was assessed by luciferase reporter assays. Decreased activity of 3’UTR-reporter construct was achieved by cotransfection with synthetic miRNAs together with target 3′UTRs. Targeted mutation of putative binding sites revealed an abrogation of miRNA-mediated suppression of luciferase activity confirming specificity of miRNA-3′UTR interaction of target genes. Here we could identify significant down-regulation of miRNA in CLL, a so far not identified cluster of deregulated miRNAs led to the identification of oncogenic transcription factors as novel target genes and pathogenic pathways in CLL. A key question remains regarding the cause of down-regulation of miRNAs mainly observed in CLL. Since no genomic hotspot is apparent, an impaired processing of pre-/pri-miRNAs or suppressive transcription factor loops have to be hypothesized and investigated to reveal the regulatory role of miRNA in malignant transformation of CLL.
Regulation of B-Cell Chronic Lymphocytic Leukaemia Cell Survival and Migration by the VEGF/SEMA3A Axis.
Authors: Murphy Philip T , Power Brendan p , Thornton Patrick D , Harmey Judith H
Published: April 2020

B-cell Chronic Lymphocytic Leukaemia (B-CLL) is characterized by the accumulation of B-CLL lymphocytes in the blood, marrow and secondary lymphoid tissues. B-CLL cells have a long survival owing to alterations in the normal pathways of apoptosis. In the marrow and lymphoid tissues CLL cells are in close contact with stromal cells that constitute distinct microenvironments. The secretion of the CXCR4 ligand, CXCL12, by stromal cells attracts B-CLL cells and provides protection from spontaneous or induced apoptosis. Studies in other cell types have shown VEGF signalling is involved in regulating CXCR4 expression levels.

The aim of this study was to examine VEGF receptor expression and the role of VEGF signalling in cell survival and CXCR4 expression. Expression levels of CXCR4 and VEGF receptors, VEGFR-1 and -2, in CLL samples were determined by flow cytometry. Expression of the VEGFR co-receptor, Neuropilin-1 (NRP1), was examined by Western blot. Cell migration towards CXCL12 was assessed using CoStar Transwell chambers (5mm pore size). Informed consent was received from all patients.

VEGFR1 and VEGFR2 positive cells in 22 patient samples ranged from 0–38% and 1.5–83% respectively. NRP1 expression was detected in all samples analysed thus far (n=6). The NRP1 ligand, SEMA3A, a competitive inhibitor of VEGF binding to NRP1, decreased CXCR4 expression in patient CLL cells (n=8, p<0.05). This decrease in CXCR4 levels correlated with reduced migration of SEMA3A treated CLL cells towards CXCL12 (88 +/− 12.7% of control levels, n=8, p<0.05). Treatment of CLL cells with the VEGFR signalling inhibitor SU5416 decreased CLL cell survival which correlated with VEGFR1 expression levels (n=16, R=0.745, p<0.01) but not with VEGFR2 expression levels.

These results show that signalling through the VEGF co-receptor NRP1 plays an important role in regulating CXCR4 levels in CLL cells, as well as CLL cell migration along a CXCL12 gradient. Our results also suggest that VEGF signalling through VEGFR1 may be particularly important in regulating CLL cell survival. Thus, the VEGFR/NRP1 signalling pathway may represent an important therapeutic target in B-CLL.
High T-Cell Lymphoma Breakpoint 1 (TCL1) Levels Are Associated with Lack of Response and Inferior Outcome Following Fludarabine, Cyclophosphamide, Rituximab (FCR) Frontline Therapy in Patients with Chronic Lymphocytic Leukemia (CLL).
Authors: Schlette Ellen , Tam Constantine S. , Herling Marco , Bueso-Ramos Carlos , Wierda William , Ferrajoli Alessandra , O’Brien Susan , Keating Michael , Jones Dan
Published: April 2020

Body: Expression of the human oncogene TCL1 in transgenic mice produces B-cell tumors that resemble human CLL suggesting a role in B-cell tumorigenesis. TCL1 protein levels in CLL vary, with the highest expression seen in the pre-germinal center subtype which is also correlated with expression of ZAP70. We compare here TCL1 and ZAP70 as markers of response and progression following FCR front-line chemotherapy in a large cohort of patients (pts.).

Methods: Paraffin-embedded pre-treatment bone marrow specimens from 243 CLL pts. who received FCR as frontline therapy between 11/99 and 01/04 were evaluated for TCL1 expression by immunohistochemistry (IHC) using a mouse monoclonal antibody developed in our laboratory. A three-tier grading system (0–2) for expression levels was used. Data on ZAP70 expression (by IHC) and IgVH mutation status was available for 195 and 76 pts., respectively. The series was evaluated for the endpoints of complete response (CR) rates, CR duration, time to failure (TTF),and overall survival (OS) as previously defined (Tam et al., Blood 2008).

Results: 54 (22%) CLL cases expressed no or minimal TCL1 protein (graded as 0), while 143 (59%) cases showed dim/partial TCL1 expression (graded as 1), and 46 (19%) cases expressed moderate to strong TCL1 levels (graded as 2; Table 1). Heterogeneous intratumoral TCL1 expression was seen in a subset of cases, especially within proliferation centers. High TCL1 expression correlated with presenting features of advanced disease, including higher pre-treatment WBC counts (p=0.01), elevated serum beta-2 microglobulin (p=0.01) and unmutated IgVH genes (p = .0007).

Most significantly, high TCL1 expression levels predicted failure to attain a CR (p=0.038), shorter TTF (p=0.0003), and inferior OS (p=0.003) following FCR therapy. Although ZAP70 and TCL1 were positively correlated (R = 0.24), TCL(−) cases were found in both the ZAP70(+) and ZAP70(−) subsets. Expression of ZAP70 was also associated with shorter TTF (p=0.004), and OS (p=0.02) but not failure to attain a CR (p=0.93) after FCR therapy.

Conclusions: In CLL pts initially treated with FCR, strong TCL1 expression by IHC, unlike ZAP70, is predictor of failure to attain a CR and is a prognostic marker of inferior outcome.

TCL1 (n=243) versus ZAP70 (n=195) expression by IHC IHC ZAP(−) (n=75) IHC ZAP(+) (n=120) TCL1 0 (n=54) 23 21 1 (n=143) 47 69 2 (n=46) 5 30 Figure Figure
Prognostic Impact of Deletions of Derivative Chromosome 9 on Patients (PTS) with Chronic Myelogenous Leukemia (CML) in Chronic Phase Treated with Nilotinib or Dasatinib.
Authors: Quintas-Cardama Alfonso , Kantarjian Hagop M. , O’Brien Susan , Borthakur Gautam , Verstovsek Srdan , Wierda William , Shan Jenny , Cortes Jorge
Published: April 2020

Background: Submicroscopic deletions of the derivative chromosome 9 [Del der(9)] mapping to regions adjacent to the translocation breakpoints occur in 9% to 15% of patients with CML. Del der(9) is a powerful prognostic indicator associated with unfavorable prognosis in patients treated with interferon-alpha (IFN-α)-based therapies. Imatinib is currently the standard treatment for patients with CML and it appears to overcome the adverse prognostic impact imparted by Del der(9).

Methods: We investigated the prognostic impact of Del der(9) in 353 patients with CML treated at our institution with the 2nd generation tyrosine kinase inhibitors (TKIs) nilotinib (n=161) or dasatinib (n=192). The presence of Del der(9) prior to 2nd generation TKIs was investigated by FISH analysis using the LSI-BCR/ABL-(ES) probe (Vysis, Downers Grove, IL) in 245 patients. The median age was 53 years (range, 15–83) and the median follow-up was 24 months (range, 1–53). The primary endpoints evaluated were complete hematologic response (CHR), cytogenetic response, and survival.

Results: Twenty-eight (11%) patients carried Del der(9) and 217 an intact der(9). Among patients with deletions, 22 were in chronic phase (CP), 4 in accelerated phase (AP), and 2 in blast phase (BP) at the start of nilotinib or dasatinib therapy. In the group of patients without deletions, 122 were in CP, 55 in AP and 40 in BP. Overall, 229 (93%) patients were assessable for response after a median of 25 months (range, 1–53) of therapy. The outcome by CML phase is shown in Table 1.

CML phase Deletion der(9) No. CCyR (%) p EFS (12 mo) p OS (12 mo) p No 122 79 0.77 86 0.05 97 0.04 CP Yes 22 75 60 78 Not done 46 No 55 36 1.0 37 0.47 60 0.95 AP Yes 4 25 67 75 Not done 27 No 40 19 1.0 0 0.85 35 0.39 BP Yes 2 0 0 0 Not done 35

There was no difference in response rates among patients in CP, but those without Del der(9) had an improved EFS and OS at 24 months compared with those carrying Del der(9) (EFS: 86% vs 60%, p=0.05; OS: 97% vs 78%; p=0.04). Notably, whereas a trend towards worse EFS (p=0.05) and OS (p=0.12) was observed in patients in CP with Del der(9) treated with nilotinib, these outcomes were not significantly affected by Del der(9) in patients receiving dasatinib (EFS: p=0.47; OS: p=0.76).

Conclusion: Our results suggest that, in contrast to what has been reported with imatinib therapy, patients with CML-CP carrying Del der(9) who failed imatinib may have a worse survival than their counterparts without deletions after treatment with 2nd generation TKI. This deleterious effect is more apparent among patients treated with nilotinib than among those receiving dasatinib.
A Whole Genome Analysis Identifies SLCO1B1 as a Determinant of Methotrexate Pharmacokinetics and Adverse Effects
Authors: Trevino Lisa R , Shimasaki Noriko , Yang Wenjian , Panetta John C. , Cheng Cheng , Pei Deqing , Chan Diana , Sparreboom Alex , Giacomini Kathleen M , Pui Ching-Hon , Evans William E. , Relling Mary V.
Published: April 2020

Methotrexate is a major component in every treatment protocol for childhood acute lymphoblastic leukemia (ALL). Both beneficial and detrimental effects of methotrexate in ALL have been clearly related to methotrexate plasma pharmacokinetics, which vary substantially among patients. However the genetic basis of such variability remains largely unknown. Herein, we surveyed 600,000 germline single nucleotide polymorphisms (SNPs) to determine how inherited genetic variation affects the disposition of methotrexate among 434 children with ALL who received 3014 courses of methotrexate at 2 to 5 g/m2. Adjusting for age, race, gender and methotrexate regimen, the most significant SNPs associated with methotrexate clearance were annotated to a plausible gene, the organic anion transporter polypeptide, SLCO1B1. The three top SNPs included rs11045879 (P = 1.7 × 10−10), rs4149081 (P = 1.7 × 10−9), and rs2900478 (P = 2.8 × 10−8). Linkage disequilibrium (LD) was observed among these three SLCO1B1 SNPs (r2=1) and with a known functional polymorphism in SLCO1B1, T521C (rs4149056, r2 = 0.86). The top two SLCO1B1 SNPs rs11045879 and rs4149081 were further validated (P = 0.018 and P = 0.017) in an independent cohort of 206 patients with ALL. Additional SNPs annotated to SLCO1B1 were identified and further validated. In a stepwise multiple linear regression analysis, SLCO1B1 genetic variation remained significant and explained clearance variability comparable to that of other non-genetic factors including treatment regimen. SNPs in SLCO1B1 were also associated with methotrexate-related gastrointestinal toxicity (P= 0.03 to 0.0005, odds ratio 8.3 to 16.4). In summary, we have identified a candidate gene, SLCO1B1, which is strongly associated with the pharmacokinetics of methotrexate, an anticancer drug with a low therapeutic index in multiple treatment regimens. Although SLCO1B1 is widely recognized as having a strong impact on the disposition of many drugs in clinical use, based on in vitro data, it was not thought to have a major role in methotrexate transport or disposition. Our study demonstrates proof of principle that genome-wide tools in clinical pharmacologic problems can lead to the discovery of important and novel pharmacogenetic links between inherited genomic variation and drug response in humans.
Efficacy of Imatinib Mesylate in the Treatment of Refractory Sclerodermatous Chronic Graft-Versus-Host Disease.
Authors: YakoubAgha Ibrahim , Magro Leonardo , Catteau Benoit , Willekens Christophe , Coiteux Valerie , Bruno Benedicte , Terriou Louis , Jouet Jean-Pierre
Published: April 2020

Outcomes in the treatment of sclerodermatous chronic graft-versus-host disease (cGVHD) are generally disappointing. Imatinib mesylate enables selective, dual inhibition of the transforming growth factor beta (TGFβ) and platelet-derived growth factor (PDGF) pathways. Recently, the drug’s effects on fibroblasts have been reported in both in vitro and in vivo studies. Inhibiting fibroblast growth (and thus decreasing collagen production in dermal fibroblasts) is thus a logical therapeutic approach. Here, we report on our experience with 12 patients who received imatinib mesylate for refractory sclerodermatous cGVHD following allogeneic stem cell transplantation (allo-SCT). The patients’ characteristics were as follows: median age, 35 years (range: 15–59); 7 male recipients, 6 female donors; 4 cases of CML, 4 MDS, 2 ALL and 2 Hodgkin’s lymphoma. The patients had received either myelo-ablative conditioning with standard GVHD prophylaxis based on cyclosporine and short-course MTX (n=9) or nonmyelo-ablative conditioning with cyclosporine and MMF. Seven patients received a marrow graft and 5 received a peripheral blood graft. All displayed refractory, chronic, sclerodermatous GHVD with at least 3 lines (range 3–6) of prior immunosuppressive therapy. The modified Rodnan skin score was used to assess the extent of skin damage. Glivec was initiated at a dose of 400 mg/day between 16 and 119 months post-transplantation (median: 44). Despite an imatinib dose reduction and the administration of various symptomatic treatments, 4 patients (33%) had to discontinue their treatment soon after its initiation (range: 16–64 days) because of intolerance (especially muscle cramps) and were not evaluable in terms of the efficacy criterion. Other side effects reported were parenthesis, diarrhea and edema. In the remaining patients, the scleroderma symptoms improved within three months of treatment initiation. At the time of this report, all patients were alive and those who tolerated imatinib mesylate have experienced a complete or near-complete response (n=4) or partial response (n=4). All responders (except for one who discontinued the drug 157 days after initiation, due to cramps) were still on the treatment, after a median time period of 216 days (range: 80–2053). This retrospective report shows that when imatinib mesylate is well tolerated, it is effective in patients with refractory sclerodermatous cGVHD and is thus a promising candidate for the treatment of this complication. This study should provide useful background information for building prospective, multicenter studies.
Long Term Outcomes for Patient with Diffuse Large B-Cell (DLBCL) or Hodgkin’s Lymphoma (HL) following Autologous Stem Cell Transplant (ASCT) Who Are Disease Free at Five Years Post Transplant.
Authors: Vose Julie M. , Loberiza Fausto , Bierman Philip , Bociek Robert , Armitage James
Published: April 2020

From 1984–2002, 294 patients (pts) with DLCL and 436 pts with HL (total 730) received high dose chemotherapy and ASCT at the University of Nebraska Medical Center. This analysis evaluates the long term outcome for pts in those cohorts who survived for five years and remained progression-free (PFS) during that interval with no further therapy. There were 92 pts with DLCL and 121 with HL who were alive and disease-free at 5 years post-transplant. The median age for pts with DLBL was 44 yrs (range 17–70) and 32 yrs (range 15–62) for pts with HL at the time of transplant. The median time to transplant from the time of diagnosis was 11 months (mo) for the DLCL group and 32 mos for HL pts. The majority of pts were chemo sensitive at the time of transplant – 80% for DLCL and 70% for HL. The median follow-up among survivors was 98 mo (range 61–222) for DLCL pts and 127 mo (range 63 – 272) for HL pts. The outcomes are as follows:

Variables DLCL HL p-value PFS @ 10 yrs 89% 82% 0.26 OS @ 10 yrs 91% 90% 0.72 Secondary Malignancies 11 (12%) 21 (17%) 0.26

Figure Figure

Cox regression analysis showed only age (> 60 yrs) at the time of transplant was associated with an increased relative risk (RR) of death; 7.64 (2.61–22.35) compared to pts < 40 yrs. Pts with DLCL had a RR of progression 0.12 (0.02–0.89) as compared to those with HL. Causes of death are as follows:

Secondary malignancy 14 (41%) Disease progression 7 (20%) Organ dysfunction 8 (24%) Infection 2 (6%) Unknown 3 (9%)

Conclusions: Patients with DLCL and HL who are progression-free at five years post-transplant have a low risk of relapse of their lymphoma. However, secondary malignancies and organ dysfunction are a long term risk from ASCT and survivors should be carefully followed for these complications.
Similar 5-Year Survival after Peripheral Blood Autotransplants (AutoPB) Versus HLA Matched Sibling Myeloablative Transplants (AlloBMT) for Acute Myeloid Leukemia (AML) in First Complete Remission (CR1).
Authors: Keating Armand , Kukreja Manisha , Silva Gisela Tunes da , Gupta Vikas , Cutler Corey , Tallman Martin S. , Sierra Jorge , DiPersio John F. , Horowitz Mary M. , Weisdorf Daniel J.
Published: April 2020

Optimum post-remission strategies for AML CR1 remain uncertain. Previous studies comparing autotransplants with alloBMT favored the latter because of less relapse despite treatment-related mortality (TRM) rates of 20–30%. In those studies, the autotransplants were exclusively with bone marrow (BM) and reported TRM of 12–20%. We were interested in determining the effect of lower TRM with AutoPB shown in many series to be 5–10%. We compared TRM, relapse, leukemia-free survival (LFS), and overall survival (OS) of 1133 patients 19–60 years of age in CR1 who underwent HLA-identical myeloablative BM or peripheral blood (PB) allogeneic transplants or AutoPB for AML reported to the CIBMTR from 1995–2004. For alloBM, alloPB and autoPB, median ages were 36, 40 and 44 yrs; cytogenetics were missing/unknown in 26%, 12% and 17%; proportion with poor risk cytogenetics was 6%, 9% and 7%; and median follow-up of survivors was 82 (6–149), 60 (4–144), and 62 (1–139) mo., respectively. Outcomes were evaluated in univariate and by multivariate analyses using the pseudo-value technique.

Results: 5 year transplant outcomes by univariate analysis are as follows:

Allo BM Allo PB Auto PB Outcomes@5years N Prob (95%CI) N Prob (95%CI) N Prob (95%CI) P-value Relapse 473 20 (17–24) 425 26 (21–30) 227 45 (38–52) <0.001 TRM 473 19 (16–23) 425 20 (17–24) 227 8 (5–12) <0.001 LFS 473 61 (56–65) 425 54 (49–59) 227 47 (40–54) 0.13 Survival 475 64 (59–68) 428 59 (54–64) 230 54 (47–60) 0.19

All analyses showed that risks associated with alloBM and AlloPB were identical, hence relative risk (RR) was modeled for any allotransplant (alloBMT) vs autoPB. Multivariate analysis of alloBMT vs autoPB cohorts revealed that age >40 yrs was associated with increased RR for TRM, LFS and OS at 5yrs:1.39 (1.05–1.84;n=518;p=0.019);1.25(1.04– 1.51;n=521;p=0.015);1.28(1.05–1.56;n=521;p= 0.012), respectively. For relapse at 5 yrs, the presence of extramedullary disease was a poor prognostic factor: [RR: 1.60 (1.13– 2.27); for no n=1036; yes n=88]. Cytogenetics risk category was not a significant covariate for TRM (p=0.84), relapse (p=0.13), LFS (p=0.27) or OS (p=0.062) at 5 yrs.

Conclusions: AutoPB may provide acceptable alternative post-remission therapy for AML CR1 in the absence of a matched sibling donor, and its role in this regard should be reinvestigated prospectively.
The Role of FDG-PET in Hodgkin Lymphoma S/P Autologous Hematopoietic Cell Transplant (HCT) as a Predictor of Outcome.
Authors: Shah Neel B. , Farhat Mohamed , Lakhani Arti , Mcfarland Daniel , Venugopal Parameswaran , Gregory Stephanie A , Shammo Jamile , Maciejewski John , Tuncer Hande , Fung Henry C
Published: April 2020

The role of PET scans in Hodgkin Lymphoma (HL), especially after autologous HCT, is undefined. Positron emission tomographic (PET) scanning has increasingly become a promising non-invasive modality for the monitoring of patients with HL. Whole body PET scans have been reported to have increased sensitivity and accuracy as compared to conventional staging methods for the initial staging of HL. The sensitivity of PET is reported as 86% compared to 37% for CT, while the specificity has been reported as 96.5% compared to 99.7% for CT. The purpose of this study was to investigate the utility of PET scanning in relapsed HL s/p autologous HCT as a prognostic indicator of transplant outcome. Here, we describe a single institution retrospective analysis of 55 patients with HL who had FDG-PET scans after stem cell transplantation.

Patients/Methods: Between 1/94 and 12/06, 55 patients with refractory or relapsed Hodgkin Lymphoma underwent an autologous HCT at our institution. They each had a FDG-PET approximately one month following stem cell transplant to evaluate for response following HCT. Patients were placed into two separate categories depending on results of the PET scan. The incidence and timing of relapse, disease free survival, and overall survival were assessed.

Results: A total of 55 patients were evaluated post autologous HCT with a median age of 32 years (ranging from 15–66) with a median follow-up of 26 months. The patients were divided into two groups based on FDG-PET activity. Twenty-nine patients had no evidence of activity post transplant, while twenty-six patients had evidence of activity. The median DFS and OS of the PET negative group were 35.8 months and 35.8 months, respectively. The median DFS and OS of the PET positive group were 18.1 months and 10.1 months, respectively. There were statistically significant differences between the two groups when comparing their DFS and OS (p=0.0035 and p=0.004), in favor of the PET negative group. There were 2 relapses in the PET negative group and 9 relapses in the PET positive group (see figures).

Conclusion: We conclude that FDG-PET scan performed after autologous HCT for patients with relapsed or refractory Hodgkin lymphoma strongly predicted transplant outcome. Patients with any significant PET uptake are at high risk of treatment failure and should be considered for additional treatment with strong contemplation for participation in clinical trials.

Figure Figure
Impact of Autologous Stem Cell Transplantation and/or Rituximab on Outcome of Patients with Relapsed Follicular Lymphoma- Retrospective Analysis of 2 Randomized Trials of the German Low Grade Lymphoma Study Group (GLSG).
Authors: Weigert Oliver , Uysal Atakan , Metzner Bernd , Pfreundschuh Michael , Schmitz Norbert , Wandt Hannes , Peschel Christian , Hoster Eva , Unterhalt Michael , Hiddemann Wolfgang , Dreyling Martin
Published: April 2020

Background: There is no standard therapeutic approach for patients (pts) with relapsed advanced stage follicular lymphoma (FL). The impact of high-dose therapy followed by autologous blood stem cell transplantation (ASCT) with or without addition of rituximab (R) to salvage regimens was evaluated.

Methods: Patients with advanced stage FL from 2 successive prospective randomized trials of the GLSG (MCP vs. CHOP and CHOP vs. R-CHOP, both followed by randomization for IFN maintenance vs. ASCT) were eligible for retrospective analysis of subsequent therapy if they had progressive, relapsed or refractory disease following 1st line treatment, were <65 years, had no prior ASCT and required treatment according to our established criteria.

Results: 222 patients were identified of whom 167 data sets were available for analysis (n=36 lost to follow-up, n=6 dead before initiation of salvage therapy, n=13 no indication for treatment). Median age was 50 years (range 19–64), 97 pts (58%) were male, 20 pts (12%) had previously received R (R-CHOP 1st line). 87 pts (53%) were treated with a R-containing salvage regimen. Most commonly used chemotherapy protocols were purin-analogue based (26%), followed by alkylator (22%) and anthracycline (13%) based regimens. Overall response rate (ORR) for chemotherapy with or without R 2nd line was 86% (CR 39%, PR 47%) and 78% (CR 26%, PR 52%), respectively. Median PFS for pts treated with or without Rituximab 2nd line was 3.4 and 2.8 years (yrs), median OS 8.3 and 7.2 yrs, respectively. Adequate numbers of peripheral blood stem cells could be harvested in 92% of evaluable patients. 66 pts received ASCT (40%) of whom 31 were also treated with R. 26 pts (39%) received a TBI-containing conditioning therapy. Most common non-hematologic grade 3/4 toxicity of ASCT were mucositis (43%) and infectious complications (30%). 4 pts died within 3 months after ASCT (6%). ORR before and after ASCT was 79% (CR 19%, PR 60%) and 95% (CR 60%, PR 35%), respectively. Median PFS for pts receiving ASCT or not was 5.5 and 2.3 yrs (p=0.038), median OS 7.2 yrs and not reached yet, respectively.

Conclusion: This retrospective analysis of two prospective multicenter trials suggests that patients with relapsed FL qualifying for ASCT may profit from that approach in terms of improved response rates and prolonged remission duration, albeit with more toxicity and an early death rate of 6%. Subgroup analysis revealed that the addition of R to salvage therapy primarily benefited patients not receiving ASCT 2nd line. Comparison of these results with ASCT in 1st remission with or without R is ongoing and will be presented at the meeting.
Significantly Reduce Acute Graft-Versus-Host Disease in Haploidentical Stem Cell Transplantation by Using Cord Blood as the Third Party Cells.
Authors: Lu Dao-Pei , Wu Tong , Gao Zhi-Yong , Wang Jing-Bo , Cao Xing-Yu , Zhao Yan-Li , Dong Lujia , Yin Yu-Ming , Ji Shu-Quan , Da Wan-Ming
Published: April 2020

More alternative donor stem cell transplantation (SCT) has been performed in recent years in China due to lack of HLA identical siblings. Higher risk for developing acute graft-versus-host disease (aGVHD) has been seen in patients who receive haploidentical stem cell transplantation (haplo-SCT) compared with identical sibling SCT patients (Lu DP et al, Blood2006; 107: 3065). In a murine model, we have first demonstrated that the animals transplanted with three mixed bone marrow (A+B+C to A) were survived longer due to milder aGVHD compared with the mice transplanted with single allogeneic bone marrow (B to A). In current clinical trial, we examine whether cord blood (CB) as the third party cells could reduce aGVHD in haplo-SCT setting under the same principle investigator in two hospitals. Between January 2006 and April 2008, total 133 haplo-SCT patients with hematological malignancies were enrolled. The patients with advanced diseases were excluded. All patients received unmanipulated blood and marrow transplant after BUCy2 or CyTBI plus antithymocyte globulin (ATG, Genzyme 10mg/kg) as preconditioning. Cyclosporine, short-term Methotrexate, and Mycophenolate mofetil were employed for GVHD prophylaxis. Fifty-six patients received one unit of HLA 3–6/6 matched CB one day before SCT as CB group, and 77 cases did not receive CB as control group. The main clinical characteristics in both groups are comparable. All patients in both groups achieved full donor chimerism. Low levels of CB chimerism were detected in a few patients at early stage after SCT. No long-term CB engraftment was found. The cumulative incidences of grade II to IV aGVHD were 16.4% versus 38.4% (p=0.008) in CB group, control group respectively. The cumulative incidences of grades III to IV aGVHD for CB group versus control group were 9.2% versus 22.4% with p=0.043. The incidences of 100-day treatment-related mortality were 1.8% versus 10.4% (p=0.053) for CB group, control group respectively. Our preliminary clinical study has shown that CB as the third party cells could significantly reduce the incidences of aGVHD, especially for severe aGVHD, and also treatment-related mortality in haplo-SCT. The mechanism of this strategy need to be further investigated.
Clinical Outcome of Aspergillus-Specific Cytotoxic T Lymphocytes for Refractory Invasive Aspergillosis in Patients with Hematological Malignancies.
Authors: Wu Tong , Tong Chun-Rong , Cao Xing-Yu , Wang Jing-Bo , Chang Lung-Ji , Liang Yin , Lu Dao-Pei
Published: April 2020

Invasive aspergillosis has high mortality in patients with hematological malignancies after chemotherapy or allogeneic stem cell transplantation (SCT) due to usually low response rate to current antifungal agents and relatively poor tolerance to the toxicities of the antifungal medicines. In this pilot clinical study, we explore to manage the patients with refractory invasive aspergillosis with aspergillus-specific cytotoxic T lymphocytes (aspergillus CTL). Ten patients who were either no longer response to antifungal medicines or have to stop antifungal agents due to severe toxicities were included in this clinical study. Diagnosis are ALL (2 cases), AML (5 cases), and CML (3 cases). The median age was 37 (10 to 62) years old. Seven of them received allogeneic SCT (unrelated 3 cases, haploidentical 3 cases, identical sibling 1 case), and 3 patients were after chemotherapy. Aspergillus CTLs were either donor origin (5 cases) or patient origin (5 cases) if original donor was not available. Patients received 1 to 3 infusions of aspergillus CTLs. The median CTLs infused were 3.61 (0.28–40.00) x 105/kg. No infusion-related reactions were noted. No pre-existed graft-versus-host disease deteriorated. With aspergillus CTL therapy, one of them (10%) achieved complete response, 6 patients (60%) improved significantly, 1 case was stable, and 2 patients became worse. Our preliminary data has shown that aspergillus-specific cytotoxic T lymphocyte is a safe and promising mean to manage refractory invasive aspergillosis in patients with hematological malignancies.