bluuebunny/crossref_metadata_2025 huggingface dataset:
Emergency Blood Supply Planning: A Statewide Prospective Audit To Determine the Potential Benefits of Triaging Red Cells during a Critical Shortage.
Authors: Shortt Jake , Polizzotto Mark N. , Waters Neil , Jones David , Borosak Marija , Moran Martine , Devine Alexandra , Jolley Damien J. , Wood Erica M.
Published: October 2019

Careful planning is critical to ensure blood availability during times of reduced supply and/or increased demand, such as infectious pandemic, act of terrorism or natural disaster. Possible strategies include triaging of supply to prioritize support where transfusion cannot be deferred. However, few data concerning urgency of transfusion are available to inform planning. We designed a random sample survey to assess urgency of clinical need, and hence potential utility of triaging red cells (RC) in an emergency through restricting use only to urgent indications, for a regional population of approximately 5 million. An audit based on random sampling of units at point of distribution was used to assess state-wide RC use over time, and capture data in proportion to actual usage over both rural and metropolitan centers. Randomly selected RC units were tagged with a case-report form during production. Tagged units were distributed to hospital laboratories with routine supply. At time of issue for transfusion, the institutional laboratory completed and returned case report forms from the tagged units. Information regarding recipient demographics, indication for transfusion, urgency of supply, and (where issued to support surgery) urgency of surgery were obtained. Following an initial pilot study to refine the model, approximately 1000 tagged RC units (46% group O, 36% A, 13% B, and 4% AB; RhD positive 85% / negative 15%) of a planned 5000 total have been introduced to the state inventory. Three percent of units were not transfused. Major clinical categories of use included hematology (17% of overall use), gastroenterology (11%), orthopedic surgery (9%), non-hematological oncology (6%), gastrointestinal surgery (6%), cardiothoracic surgery (5%), obstetrics/gynecology (5%), urology (3%), trauma (3%) and pediatrics (2%); miscellaneous indications and anemia not otherwise specified constituted 10% and 22% of transfusions respectively. 18% of units were used in acute transfusion episodes (needed within 1h), 43% for urgent indications (1–24h), 33% semi-urgent (24h - 1 week) and 4% non-urgent. Of the 34% units allocated to support perioperative bleeding; only a quarter of procedures were elective. In 15% of cases the urgency of surgery was not stated. Thus, reporting institutions indicated that only 38% transfused units could have been deferred for more than 24h, and only 9% of total RCs were allocated to support potentially deferrable elective surgery. These early results suggest that triaging of RC supply in an acute shortage would only have a very short term impact on actual RC use, as the majority of RC use is already relatively urgent. Additional strategies for emergency blood contingency planning are required to meet priority clinical needs.
Polymorphic Variation at the STAT5 Locus Is Associated with the Response of CML Patients to Interferon Alpha.
Authors: Kreil Sebastian , Waghorn Katherine , Hehlmann Rudiger , Reiter Andreas , Hochhaus Andreas , Cross Nicholas C.P.
Published: October 2019

Interferon alpha (IFN) induces complete hematologic remission in 70% to 80% of patients with CML in early chronic phase, with up to 8–30% of cases achieving a complete cytogenetic response (CCR). Although patient responses correlate with Hasford and Sokal risk scores, the molecular basis for this heterogeneity remains poorly understood. We hypothesized that polymorphic differences in the IFN signal transduction cascade might account for different patient responses. IFN binds the heterodimeric type I interferon receptor, the two subunits of which are encoded by IFNAR1 and IFNAR2. Binding activates the JAK1 and TYK2 non-receptor tyrosine kinases, which phosphorylate and activate STAT proteins. We studied 187 cases treated with IFN based regimens as part of the German CML studies I-III. Of these, 105 were defined as non-responders (no cytogenetic response) and 82 as responders (achievement of <=34% Philadelphia-chromosome positive metaphases) after a median treatment duration of 23 and 40 months, respectively. There was no significant difference in the incidence of der(9) deletions between the two groups. We genotyped 14 single nucleotide polymorphisms (SNPs) within or close to the genes encoding IFNAR1, IFNAR2, JAK1, TYK2, STAT1, STAT3 and STAT5a/b and compared allele frequencies between responders and non-responders. Significant differences were found for two SNPs at the STAT5a and STAT5b loci. For rs16967611 the minor allele frequency (maf) was 0.34 for non-responders and 0.28 for responders (P=0.21; chi-squared test) and for rs6503691 the maf was 0.16 for non-responders and 0.07 for responders (P=0.005). The latter SNP remained significant when corrected for multiple testing. Three other SNPs at STAT5a/b (rs9900213, rs17500235 and rs17591972) were also analyzed and yielded P values of 0.87, 0.01 and 0.15 respectively. STAT5a, STAT5b and STAT3 are tightly linked at 17q21 and show strong linkage disequilibrium according to HapMap data. To determine if the genotype at rs6503691 is related to gene expression levels, we measured STAT5a, STAT5b and STAT3 mRNA in 214 pretreatment CML patients by real time quantitative PCR. There was no difference in the median normlised expression of STAT3 and STAT5a in cases with a c/c genotype compared to those with c/t or t/t, however for STAT5b the expression level was higher in cases with a c/c genotype (P=0.02). We conclude that rs6503691, located within STAT5b intron 1, is associated with the expression level of STAT5b and response of CML patients to IFN.
The Expression of SALL4, a Newly Identified Stem Cell Factor, Is Correlated with Chronic Myeloid Leukemia (CML) Disease Progression.
Authors: Ma Yupo , Yang Jianchang , Kong Nikki , Amin Hesham M. , Chai Li
Published: October 2019

The homeotic gene, SALL4, plays an essential role in the maintenance of embryonic stem cell pluripotent and self-renewal properties by interacting with two other key regulators in ESCs, Nanog and Oct4. Previously, we have demonstrated that SALL4 contributes to acute myeloid leukemia (AML) development in a murine model. Also, Bmi-1, a key regulator in leukemic stem cells (LSCs), is a direct target of SALL4. In this study, through immunohistochemistry and fluorescent activated cell sorting (FACS) analysis, we demonstrated that the expression level of the SALL4 protein correlated well with disease progression in CML. There was no SALL4 positive population detectable in normal whole bone marrow samples or chronic phase CML samples. A distinguish SALL4 positive population was present in accelerated and blastic phases of CML marrows, and overlapped with a subset of CD34+ population. This finding is particular of interesting since committed Granulocyte-Macrophage Progenitors (GMPs) are CD34+/CD38+ cells and have been proposed to be candidates for LSCs in the AML to CML transformation. Decreasing SALL4 expression in a CML cell line (KBM5) down regulated Bmi-1 expression, which resulted in increased caspase-3 activity, induced cell-cycle arrest, and decreased cell proliferation. Furthermore, restoration of Bmi-1 expression rescued apoptosis, increased cell proliferation, and promoted cell-cycle progression. Our data have extended our understanding on the stem cell factor SALL4 in CML transformation and its role in the pathogenesis of CML disease progression. SALL4 can also potentially be used as a novel diagnostic marker for CML transformation.
A Prospective Gene Expression Profiling Study for Prediction of Cytogenetic Response in Early and Late Chronic Phase CML.
Authors: Wolf Dominik , Schmidt Stefan U. , Fong Dominik , Wrulich Oliver , Lion Thomas , Überall Florian , Petzer Andreas , Gastl Günther
Published: October 2019

Gene expression profiling has proven useful for identification of gene sets predicting response and/or prognosis in various hematological malignancies. So far expression profiling studies in CML predicting response to imatinib therapy suffered from major flaws, as previous retrospective studies relied on various technical platforms using rather small patient populations. Here, we present the interim results of an expression profiling study performed within a prospective multicenter CML trial of the Central European Leukemia Study Group(CELSG) comparing standard (SD) with high-dose (HD) imatinib as second line treatment for chronic phase CML. Blood samples from at total of 102 CML patients with early or late chronic phase disease were collected prior to and six weeks after treatment with either 400 mg/day for 12 months or 800 mg/day for 6 months followed by 6 months of 400 mg imatinib therapy. Whole blood samples preserved by PAX gene technology were further processed and evaluated centrally at the expression profiling core facility at Innsbruck Medical University. For expression profiling samples were subjected to hemoglobin mRNA reduction before target preparation for hybridization to Affymetrix hGU133 Plus 2 genechips detecting ∼47000 transcripts thereby allowing a whole genome profiling approach. For response prediction we used both the linear discriminatory analysis (LDA) and a random Forest (RF) decision tree algorithm provided in Bioconductor software packages for the open source statistical language “R”. A 100- or 10-fold crossvalidation was performed using a robust leave-10-out setting for LDA and RF respectively. The planned interim analysis of clinical data from 76 evaluable patients undergoing 12 months of therapy with daily doses of 400 mg or 800 mg imatinib revealed 50 (66%) patients with and 26 (34%) patients without a major cytogenetic response (<35% Ph+ metaphases). We found, irrespective of imatinib dosing, a predictive gene expression signature for achieving a major cytogenetic response after 12 months of imatinib therapy. This predictive gene set comprises five genes (ENTPD4, F5, SCL22A4, AIM2, TLR5, PXK) achieving a correct prediction rate of 87% within the 6-week-treatment patient subgroup and an overall prediction rate of 76%. In conclusion, this is the first prospective gene expression profiling study in CML indicating a gene expression profile predicting major cytogenetic response, which still represents a robust response parameter for long-term outcome during imatinib therapy of patients with early or late chronic phase CML.
Philadelphia Negative BCR/ABL1 Positive CML: FISH Mapping and aCGH Reveal Breakpoint Hotspots.
Authors: Virgili Anna , Brazma Diana , Grace Colin , Chanalaris Anastasios , Reid Alistair , Apperley Jane F. , Nacheva Elisabeth P.
Published: October 2019

Chronic myeloid leukaemia (CML) is a pluripotent haematopoietic stem cell disorder defined by expression of the BCR/ABL1 fusion gene, a constitutively activated tyrosine kinase. The fusion gene is a result of t(9;22)(q34;q11) or related variant rearrangements leading to formation of the Philadelphia chromosome (Ph). In less than 2% of the CML patients the bone marrow cells appear to have a normal karyogram although the fusion gene can be identified by molecular means and located by FISH on 22q11 and less frequently on 9q34 or on a third chromosome. The biology and clinical significance of the genetic rearrangements in Ph negative BCR/ABL1 positive disease are controversial and widely discussed. Two alternative mechanisms have been postulated but not confirmed: simple insertion or multiple step model. Although sequencing analysis as early as 1990 (Morris and Heisterkamp) showed that a large part of the 9q34 region is present in the masked Ph, little has been done to clarify the underlying rearrangements. Aided by genome array CGH and FISH, we constructed a comprehensive map of the regions flanking ABL1 and BCR genes in 9 patients and one cell line with this form of CML. The main results show:

the formation of the BCR/ABL1 fusion is associated with relocation of sequences well in excess of the ABL1 and BCR gene in all 10 samples, the involvement of downstream 3′ ABL1 is diversely associated with distal breakpoint clusters in the regions of the RAP-GEF1 and RXRA genes, the disease progression can be accompanied by sequential breakages/reunions within the regions flanking the BCR/ABL1 fusion and the presence of multiple cryptic rearrangements of both 9q34 and 22q11 segments identifies a sub-group of patients with shorter survival thus demonstrating the clinical significance of genome instability in Ph negative, BCR/ABL1 positive CML.
MicroRNAs as the Target of Deletions on der(9) in Chronic Myeloid Leukemia.
Authors: Albano Francesco , Anelli Luisa , Zagaria Antonella , Pannunzio Alessandra , Rossi Antonella Russo , Manodoro Floriana , Liso Vincenzo , Rocchi Mariano , Specchia Giorgina
Published: October 2019

Deletions on der(9) are associated with chronic myeloid leukemia(CML) in 15–18% of cases. To date, the biological significance of this genomic loss in the pathogenesis of CML is unknown. The most plausible hypothesis is that the loss of a tumor suppressor gene may confer a proliferative advantage to the Philadelphia-positive clone. On the other hand, it has now become evident that microRNAs (miRNAs) play an important regulatory role in some hematological malignancies. To investigate the presence of miRNAs within the genomic regions lost on der(9) we analyzed 60 CML patients with der(9) deletions. Methods. Genomic characterization of the deleted sequences was performed by fluorescence in situ hybridization (FISH) using a contig of DNA clones; the miRBase (http://microrna.sanger.ac.uk/) was queried to assess the presence of miRNAs in the der(9) deleted genomic regions. FISH experiments showed that the genomic loss on der(9) of the 9 (centromeric to ABL) and 22 (telomeric to BCR) chromosome sequences ranged from 260 Kb to 54 Mb and from 230 Kb to 12.9 Mb, respectively. Consultation of the miRBase revealed that in 16 (27%) patients there was loss of miRNAs mapping on chromosome 9 whereas no known miRNAs were mapped on the deleted genomic sequences belonging to chromosome 22. Moreover, 4 cases with a complex t(9;22) rearrangement and der(9) deletions showed loss of the miRNAs sequence also on the third derivative chromosome (4p16, 7p14, 13q14, and 11q13, respectively); among them, only in one case the loss of miRNAs on the third derivative was not associated with the miRNAs deletion mapped on chromosome 9. The most recurrent miRNAs deleted on der(9) were mir-219–2 (deleted in 100% of cases) and mir-199-b (lost in 67% of cases). It is noteworthy that mir-219–2 neighbors and overlaps CpG-islands, suggesting a potential role of this miRNA in CpG-island methylation. Experimental studies indicate that miRNAs can function as tumor suppressor genes or as oncogenes. In fact, in chronic lymphocytic leukemia associated with del(13)(q14) it has been demonstrated that the miRNAs loss can induce downregulation of the antiapoptotic BCL-2 protein. The novel evidence that deletions on der(9) in CML are associated with miRNAs loss may shed new light on the significance of genomic sequences loss. Further studies are needed since it is known that some microRNAs may have as many as a few thousand targets, so prediction algorithms and strategies allowing large-scale screening of multiple target genes are required.
NM23-H2 Is a Tumor Associated Antigen (TAA) in Chronic Myeloid Leukemia (CML).
Authors: Tschiedel Sabine , Jilo Annette , Niederwieser Dietger W.
Published: October 2019

Introduction: The human Nm23 family consists of 8 genes, termed Nm23-H1 through -H8. H1 and H2 are 88% identical and localized to chromosome 17. Both Nm23-H1 and -H2 display nucleotide-diphosphate kinase (NDPk) activity responsible for balancing NTP levels by transferring gamma-phosphates between nucleotides, and individually demonstrate a variety of other activities including DNA binding, transcriptional regulation and apoptotic cleavage of DNA. A variety of studies imply a role for Nm23 proteins in development and in a range of cancers, in which level of expression rather than mutation correlates to prognosis. Specifically, a mutual transcriptional activation between the nm23-H2 and c-myc genes has been implicated in transformation. Our group described the identification of Nm23-H2 as a candidate tumor-associated antigen in a case of CML and demonstrated the presence of Nm23-H2 reactive T cells 5 years after transplantation. Here, we compare the expression of Nm23-H2 and H1 proteins in leukaemic patients and healthy donors.

Materials and Methods: PBMC were collected from 10 CML patients at initial diagnosis, from 4 Glivec-resistant CML patients, from 12 AML patients at diagnosis and from 5 healthy donors. PBMC, CD3+, CD14+, CD33+ and CD34+ populations were stained, fixed and sorted by FACS onto microscope slides. Immunocytochemical analysis was performed with Nm23-H1 (Novacastra), Nm23-H2 (Santa Cruz) and β-Actin (Sigma) specific antibodies. Evaluation was carried out using a confocal laser scanning microscope LSM 510 (Zeiss).

Results: PBMC from 9 out of 10 untreated CML patients (90%) showed a strong Nm23-H2 staining. Fractionation of the PBMC population revealed that CD14+, CD33+ and CD34+ but not CD3+ cells are Nm23-H2 positive in the majority of these patients. Nm23-H2 was also detected in the PBMC of 3 out of 4 Glivec-resistant CML patients. In most cases, Nm23-H2 co-localized with β-actin. Under the same staining conditions, PBMC and sorted subpopulations from 11 of 12 AML patients (92%) and from all healthy donors were Nm23-H2 negative, while very weak staining was detected in PBMC of one AML patient. Nm23-H1 protein was clearly detectable in a number of cell lines (HL-60, U-937, K562 and T47D), but was very weak or absent in PBMC from CML patients and healthy donors.

Conclusion: Taken together, our results show that those myeloid populations functionally affected by the bcr/abl mutation specifically over express Nm23-H2 but not Nm23-H1 protein in 9 out of 10 cases of CML. While the molecular basis of this expression pattern and the possible involvement of c-Myc remains to be resolved, these results support the proposal that immune responses to Nm23-H2 may mediate GvL effects and that peptides derived from Nm23-H2 might therefore be attractive targets for specific immunotherapy in CML patients.
Immune Effects of Imatinib in Chronic Myelogenous Leukemia In Vitro.
Authors: Bund Dagmar , Buhmann Raymund , Kolb Hans-Jochem
Published: October 2019

Imatinib mesylate, a potent and selective inhibitor of the BCR-ABL tyrosine kinase, has been shown to induce durable haematological and major cytogenetic responses in a high percentage of CML patients. However in most patients the disease recurs, when imatinib is discontinued. In contrast, allogeneic stem cell transplantation (ASCT) is considered to be curative by the immune effect of donor T cells against CML progenitor cells. In this context, the role of imatinib is controversial; it may improve the results of ASCT by reducing the tumour load, it may also reduce the effect of donor lymphocyte transfusions (DLT) by impairing the function of T cells and the capacity of myeloid cells to present antigen. Patient derived CML-cells were studied for the stimulation of allogeneic HLA-matched and mismatched T-cells in the presence and absence of imatinib. In a 5 day culture the proliferative response of HLA-mismatched T cells was evaluated in presence of different concentrations of imatinib (0, 1, 2, or 5 micro M) and various responder-to-stimulator ratios. Thereby, proliferation was detected via a CFDA based assays and the activation profile (CD25, CD69) of the T-cells was determined by FACS. Cr51-release assays were performed after a 7 day culture of CML cells with HLA-matched T cells to test cytotoxicity of CD8+ T-cells. In addition, we characterized the antigen-presenting profile (CD14, CD33, HLA-DR, CD40, CD80, CD86, CD54, CD58) of the CML cells over a 5 day culture with and without imatinib. The presence of imatinib inhibited the proliferative capacity of allogeneic T-cells in a dose-dependent manner. Also, the expression of T cell activation markers was reduced in the presence of the different imatinib concentrations. Preincubation of CML cells with imatinib for 48 hours strengthened the effect on proliferation and activation of T cells. Moreover, imatinib impaired the cytotoxic function of T-cell (HLA-matched setting; CR51-release assay) also in a dose-dependent manner. Finally, the antigen-presenting profile of the myeloid leukemia cells was down regulated by increasing concentrations of imatinib. In summary, imatinib may interfere with the T cellular immune response and the antigen presenting profile on the CML cells in vitro. These results may have an impact on new strategies of treatment of CML with immunotherapy.
Efficacy of Imatinib Mesylate Dose-Escalation in CML Patients Showing Suboptimal Response to Standard Dose.
Authors: Kim D.Y. , Kim H.J. , Jeong J.S. , Kim S.J. , Joo Y.D. , Sohn S.K. , Kim I. , Kim J.S. , Song H.S. , Yoon S.S. , Kim C.S. , Jung C.W. , Yi K.H. , Min Y.H. , Hyun M.S. , Lee N.R. , Chang D.Y. , Jo D.Y. , Kim J.A. , Park J.W. , Kim B.K. , Park S.
Published: October 2019

Introduction: We report the results of our prospective single arm study performed to evaluate the efficacy of Imatinib mesylate (IMT) dose-escalation therapy and the influence of BCR/ABL mutational status on the treatment outcome.

Methods: Patients with chronic phase(CP) chronic myelogenous leukemia (CML) who had not achieved a complete hematologic response (CHR) after 3 months, a major cytogenetic response (MCyR) after 6 months, nor a complete cytogenetic response (CCyR) after 6-12 months of standard IMT therapy (400mg per day) were included. Patients in accelerated phase (AP) or blast crisis (BC) who had failed to achieve CHR after 3 months of IMT (400 or 600mg per day) were also eligible. 600mg of IMT had been administered for patients with CP CML per day, and 600mg or 800mg for AP or BC CML for at least 12 months. For each patient, BCR/ABL gene mutation test was performed on 6 loci. Cytogenetic response (CyR) was evaluated with the percentage of bcr/abl positive cells in bone marrow aspirate by means of FISH per 6 months. Molecular responses (MR) were assessed per 3 months with a logarithmic decrease in BCR-ABL/ABL gene ratio (BA/A) of peripheral blood or bone marrow aspirate from a standardized median value of BA/A in 56 patients with newly diagnosed CML.

Results: 77 patients were enrolled and 61 patients were assessable for the evaluation of MR. The median age was 50 years old (range: 20-70). Stages at the time of the enrollment were CP for 56, AP for 4, BC for 1 patient(s). The median time from the initiation of IMT therapy to dose-up was 20 months (range: 0-62). BCR/ABL gene mutational status could be obtained in 24 patients (wild:mutant=20:4). The median follow-up was 19.9 months (range: 6.5-24.3). For 34 patients whose 6th-month CyR is available, MCyR rate (MCyRR) was 26.5% and CCyR rate (CCyRR) was 23.5%. The 12th-month MCyRR was 61.1% and CCyRR was 50.0%. Patients with wild type showed a MCyRR of 20% and a CCyRR of 15% on the 12th month after dose-up, whereas no patient with mutant type showed a CCyR or a MCyR. CMR rate and MMR rate on the 12th-month were 16.7% and 26.7%. Among 20 patients with wild type, one patient achieved a 6th-month CMR, 2 patients MMR’s, whereas no patient with mutant type could achieve a MMR during the entire treatment period. Hematologic and non-hematologic toxicities were tolerable.

Conclusion: IMT dose-escalation could induce quite a number of durable CyR’s and MR’s without any serious toxicity in patients who had failed to achieve either a CHR or a CyR with a standard IMT therapy.
Human Bone Marrow as a Source of Multifunctional CMV-Specific CD4+ T Cells for Adoptive Cell Therapy.
Authors: Na Il-Kang , Letsch Anne , Noack Ines , Bauer Sandra , Geginat Jens , Reinke Petra , Volk Hans Dieter , Thiel Eckhard , Scheibenbogen Carmen
Published: October 2019

Introduction Adoptive cell transfer of ex vivo primed and expanded human cytotoxic T lymphocytes (CTLs) has emerged as a promising approach to treat both infectious and malignant diseases in humans. First clinical studies have shown that transfer of Cytomegalovirus (CMV)-specific CD8+ T cells is safe and effective in reconstitution of cellular immunity against CMV disease. Efficacy of adoptive T cell therapy is limited by the numbers of CTLs in vitro and the survival and function after infusion. CD4+ T cells may enhance activity via direct or indirect effector functions (Matloubian 1994 [1]). In this study we have analysed whether bone marrow is superior to peripheral blood for expansion of CMV-specific T cells. Experimental design Paired peripheral blood and bone marrow samples were obtained from patients who underwent total hip arthroplasty. By using two different protocols T cells were expanded in the presence of IL-2 and IL-7 either from bulk culture with exposure of two different peptide pools (IE1 and pp65) or after selection via IFN-γ secretion by stimulation with pp65. CMV specific immune responses were assessed by using multiparameter flow cytometry staining cells for CD3, CD4, CD8, CCR7 and CD45RA and for the cytokines IFN-γ IL-2 and TNF at day 0 and after 10 days of in vitro expansion.

Results Similar frequencies of cytokine-producing pp65– and IE1-specific CD4+ and CD8+ T cells were found in unmanipulated paired PB and BM samples. Expansion of CMV-specific T cells from BM resulted in significantly higher frequencies of specific CD4+ T cells than from PB, whereas no difference in frequencies of CMV-specific CD8+ T cells was observed. Interestingly, significantly higher frequencies of BM pp65 and IE1-specific CD4+ T cells were multifunctional, characterized by producing simultaneously IFN-γ, TNF and IL-2 (IE1: BM mean 0.44% ± 0.16; PB mean 0.09% ± 0.05, p=0.031; pp65: BM mean 3.87% ± 2.46; PB mean 1.24% ± 0.90, p=0.031). Expansion of multi-functional CD4+ T cells from BM was observed with both the bulk and selection assay protocol. Both PB and BM CMV-specific CD4+ and CD8+ T cell lines had a predominant CD45RA-CCR7- effector memory phenotype.

Conclusions This study implicates the use of human bone marrow as a source for expansion of multifunctional CMV-specific CD4+ T cells. Recent studies in HIV and Leishmania support the crucial role of multifunctional T cells in disease control (Darrah 2007 [2]).
Iron Overload Adversely Affects Survival and Increases Risk of Graft-Versus-Host Disease and Blood Stream Infections after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Pullarkat Vinod , Blanchard Suzette , Tegtmeier Bernard , Dagis Andrew , Patane Kathryn , Ito James , Forman Stephen J.
Published: October 2019

Background: Iron overload, mainly from multiple red cell transfusions is common in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) for hematologic disorders. Although the adverse impact of iron overload on survival of patients undergoing HSCT for thalassemia are well established, the impact of iron overload on the transplant outcome and complications in non-thalassemic patients is less clear.

Methods: Serum ferritin level, a marker of tissue iron overload was measured immediately prior to initiation of transplant conditioning in adult patients undergoing myeloablative HSCT from matched sibling or unrelated donors. The effect of elevated pretransplant ferritin (defined as ferritin over 1000ng/ml) on day 100 mortality, acute graft-versus-host disease (GVHD), infectious complications were assessed.

Results: Data on 190 patients were analyzed for day 100 mortality, overall survival, the combined endpoint of incidence of acute GVHD (Grade II or above) or death within 100 days and the combined endpoint of blood stream infection or death within 100 days. In univariate analysis, the high ferritin group had increased day 100 mortality (20% vs 9%, P=0.04), decreased overall survival (log-rank test: P-value=0.004), increased acute GVHD/death (63% vs 43%, P=0.009) and increased incidence of blood stream infections/death (60% vs 44%, P=0.04). In a multivariate analysis, high ferritin was associated with increased risk of death (Cox Model: hazard ratio 2.28, P-value=0.004), increased day 100 mortality (generalized linear models (GLM) odds ratio 3.82, P-value=0.013), increased incidence of acute GVHD/death (GLM odds ratio 3.11, P-value=0.001) and increased risk of blood stream infections/death (GLM odds ratio 1.99, P-value=0.032). The results remained similar when serum ferritin was considered a continuous variable.

Conclusions: Elevated serum ferritin adversely impacts day 100 mortality and overall survival after allogeneic HSCT. In addition, high serum ferritin also increases the likelihood of acute GVHD and blood stream infections after allogeneic HSCT.
The Mask Mutation Identifies TMPRSS6 as an Essential Suppressor of Hepcidin Gene Expression, Required for Normal Uptake of Dietary Iron.
Authors: Beutler Ernest , Lee Pauline , Gelbart Terri , Du Xin , Beutler Bruce
Published: October 2019

Hepcidin, the central negative regulator of iron absorption and iron release from macrophages, is upregulated by iron. Mutations in hemojuvelin, Hfe, transferrin receptor 2, and SMAD4 are known to prevent upregulation. Additionally, the bone morphogenetic proteins (BMPs), and the inflammatory cytokines IL-1 and IL-6 stimulate hepcidin gene activation. Downregulation of hepcidin is effected by anemia and hypoxia, but nothing is known of the mechanism through which this occurs. Here we describe the recessive ENU-induced phenotype Mask, so called because affected homozygotes developed regional alopecia in which truncal hair was shed while facial hair was retained. The Mask phenotype was found to be a manifestation of iron deficiency, and was eliminated by correcting the iron deficiency. When fed an iron deficient diet, mutant mice absorbed less iron than controls, as measured by total body 59Fe counting. After reaching a plateau total body counts stabilized, indicating that blood loss did not play a role in the iron deficiency. The level of liver hepcidin mRNA of iron deficient mice is normally greatly decreased; in contrast, the Mask mouse had high liver hepcidin mRNA levels. By positional cloning, we were able to ascribe the Mask phenotype to a splicing error in the Tmprss6 gene, which encodes a membrane-bound serine protease of previously unknown function. The mutation truncates the protein, eliminating the serine protease domain. Transfecting HepG2 cells to express the wildtype TMPRSS6 protein decreased baseline hepcidin reporter activity and almost entirely blunted the hepcidin inducing effect of IL-6, IL-1, hemojuvelin, and the BMPs. A construct encoding the Mask truncation mutant had diminished activity. Thus, TMPRSS6 powerfully down-regulates hepcidin gene transcription in the baseline state and prevents its upregulation by all known stimulators. TMPRSS6 is a non-redundant component of a hepcidin suppression pathway that exerts dominant effect over all known hepcidin inducing pathways, and is required for normal absorption of dietary iron.
Hematopoietic Stem Cell Transplantation (HSCT) in Waldenström Macroglobulinemia (Wm), Update of the French Experience in 54 Cases.
Authors: Dhedin N. , Tabrizi R. , Bulabois P.E. , Le Gouill S. , Coiteux V. , Dartigeas C. , Robin M. , Huynh A. , Larosa F. , Cacheux V. , Garnier A. , Morel P. , Kuentz M. , Dreyfus B. , Desablens B. , Guillerm G. , Pignon B. , Rio B. , Bilger K. , Cabrespine A. , Uzunov M. , Bay Jacques-Olivier , Leblond V. , Tournihac O.
Published: October 2019

Introduction: HSCT has been developed in few Wm cases and is nowadays challenged by other innovative approaches. However, high dose therapy followed by autologous HSCT (HD-auto) produces high response rate and some long term responses while allogeneic HSCT performed after either myeloablative (MA-allo) or reduced intensity conditioning (RIC-allo) regimens may be cure of Wm (Dreger 1998, Tournilhac 2003, Maloney 2006).

Methods: We updated and extended our retrospective experience on 32 HD-auto, 11 MA-allo and 11-RIC-allo performed from 1990 to 2006 in 51 patients from 18 institutions. A MA-allo and a RIC-allo were performed in 1 and 2 cases respectively following relapse after a 1st HD-auto.

Results: Data are presented in the table.

HD-auto MA-allo RIC-allo Nb 32 11 11 Median age at transplant 56 46 56 Interval: diagnosis-transplant 38 50 74 Chemoresistance at transplant 25% 36% 55% Conditioning regimen BEAM (13), TBI/melphalan (9), TBI/endoxan (7), other (3) TBI/endoxan (9), other (2) TBI/fluda (10), other (1) Donor Sibling (9), unrelated (2) Sibling (8), unrelated (2), cord blood (1) Median follow up (m) 45 (3–121) 68 (3–132) 22(2–60) Relapse 18 (56%) 4 (36%) 0 Transplant related mortality 12,5% (one 2th cancer) 36% 27% (one 2th cancer) overall survival (1;3;5y) 87%, 77%, 58% 64%, 54%, 54% 82%, 68%, 68% Event free survival (5y) 25% 48% 68%

Acute GVHD developed following 9 MA-allo [Grade III-IV (n=1)] and 8 RIC-allo [Grade III-IV (n=1)]. Chronic GVHD developed following 7 MA-allo [limited (n=5), extensive (n=2)] and 5 RIC-allo [limited (n=2), extensive (n=3)].

Conclusion: We confirm that autologous HSCT achieves some long term responses even in heavily pretreated patients. Allogeneic HSCT induces very long term disease control and may cure WM. Specially, the RIC-allo gives impressive results on disease control in a set of older patients, with refractory disease, mostly heavily pretreated.
Allogeneic HSC Transplantation for Multiple Myeloma: A Single Institution Experience.
Authors: Costa Luciano J. , Kumar Shaji , Dispenzieri Angela , Hayman Suzanne E. , Buadi Francis K. , Litzow Mark R. , Gertz Morrie A. , Lacy Martha Q.
Published: October 2019

Background: Despite development of new therapies and advances in autologous hematopoietic stem cell transplantation (HSCT), MM remains, for the most part, an incurable condition. Allogeneic HSCT, through induction of a graft-versus-myeloma effect, can potentially overcome resistance to conventional therapy and induce prolonged disease control. Such approach is limited by the risk of acute and chronic GVHD and the high treatment-related mortality and has been restricted to young patients with aggressive or refractory disease. We reviewed our experience with allogeneic HSCT at Mayo Clinic, Rochester, MN.

Methods: Retrospective single-institution review of consecutive allogeneic transplants performed for multiple myeloma between 1991 and 2006.

Results: Thirty-three patients underwent a myeloablative (52%) or reduced-intensity conditioning (48%) HSCT 3.2 months to 15 years after their diagnosis of multiple myeloma (n=31) or plasma cell leukemia (n=2). Median age at the time of HSCT was 48 years (range 33–61) and 19 patients (57%) had received one (n=12) or two (n=7) prior autologous HSCT. Donors were HLA-matched siblings in 25 cases (76%), matched unrelated (n=8), and 5/6 (n=1) and 6/6 (n=1) match, non-sibling family members. Source of HSC was peripheral blood exclusively in 21 (64%) and bone marrow in 12 (35%) transplants. Outcomes are being reported after a median follow up of 21.8 months (25.6 for survivors). Day+100 mortality was 21.2% and the incidences of any grade and grades 3/4 acute GVHD were 54.5% and 33.3% respectively. Among the 26 patients surviving beyond day+100, 57.7% developed chronic extensive GVHD. All 6 patients transplanted after a complete response (CR) to prior therapy remained so after the transplant. Of the 27 patients with measurable disease, disease reassessment could be performed for 20 after the transplant. Ten patients (50%) achieved a CR or near-CR and 8 patients (40%) obtained a partial response. Median time to progression and overall survival (Figure) after HSCT were 21.8 and 40.6 months respectively. Twenty-one patients have died, 10 as a consequence of disease progression. Twelve patients are alive, including 6 patients in CR, 8.3 to 172.7 months after the transplant. In a multivariate model the use of a non-sibling donor and peripheral blood HSC were associated with decreases risk of relapse, but had no effect on overall survival.

Conclusion: Allogeneic HSCT is a feasible treatment for young patient with adverse disease features. Graft-versus-myeloma can induced myeloma eradication and prolonged disease control.

Figure Figure
Efficacy of Allogeneic Stem-Cell Transplantation for T/NK-Cell Lymphomas in Adults.
Authors: Le Gouill Steven , Milpied Noel-Jean , Buzyn Agnes , de la Tour Regis Peffault , Vernant Jean-Paul , Mothy Mohamad , Ifrah Norbert , Bouabdallah Krimo , Bulabois Claude-Eric , Dupuis Jehan , Rio Bernard , Attal Michel , Yakoub-Aga Ibrahim , Tournilhac Olivier , Gratecos Nicole , Blaise Didier , Volteau Christelle , Michallet Mauricette
Published: October 2019

Mature peripheral T/Natural killer (NK)-cell neoplasms represent 10–15% of non-Hodgkin’s lymphoma (NHL) in adults. T/NK-NHL have a worst prognosis compared with B-cell lymphomas. Allogeneic stem cell transplantation (allo-SCT) is an attractive option for these patients. On behalf of the SFGM-TC group, we conducted a retrospective analysis and included seventy-seven T/NK-cell lymphoma patients. Diagnosis were: ALCL (n=27), Peripheral T-cell Lymphoma Not-Otherwise Specified (PTCL-NOS) (n=27), Angioimmunoblastic T-cell Lymphoma (AITL) (n= 11), Hepatosplenic g/d lymphoma (HSL) (n=3), T-cell granular lymphocytic leukemia (T-GLL) (n=1), nasal NK/T-cell lymphoma (nasal-NK/L) (n=3) case or non-nasal NK/T-cell lymphoma (non nasal-NK/L) (n=2), enteropathy-Type T-cell (n=1) and HTLV-1 lymphoma (n=2). Fifty-seven patients received myeloablative conditioning regimen prior allo-SCT. Donors were HLA-matched in 70 cases and related in 60 cases. Patients status at the time of allo-SCT was CR in 31 cases, PR in 26 cases and SD/PD in other cases. Five-year toxicity-related mortality (TRM) rate was 34%. Major cause of death was infection. Five-year OS and EFS rates were 57% and 53.3%, respectively. In a multivariate analysis, chemoresistance disease (SD, refractory or progressing disease at the time of allo-SCT and aGVHD grade III/IV were the only adverse prognostic factors for OS (p= 0.027 and p=0.033, respectively). Disease status at transplantation influenced EFS (p= 0.0032) and a HLA-mismatched donor increased TRM (p= 0.0386). A plateau was reached after one and a half year after allo-SCT. Only 5 out of 59 patients in CR after allo-SCT experienced a relapse. The 5-year OS rate for chemo-resistant patients was also encouraging. These patients were not curable with conventional approaches and near of one third have taken advantage of allo-SCT. Furthermore, two patients received DLI at relapse and they both reached a second durable CR. Taken together, this suggests that there is a graft versus T-/NK-lymphoma effect which may play a role in the curative potential of allo-SCT. we conclude that randomized clinical trials comparing allo-SCT versus conventional chemotherapy upfront for PTCL, aggressive AITL or histopathological subtypes (HSL, HTLV-1 lymphomas) have to be encouraged.
Effect of HLA-Matching on Outcome of Hematopoietic Stem Cell Transplantation from Unrelated Donors: A IBMDR-GITMO Study.
Authors: Crocchiolo Roberto , Ciceri Fabio , Oneto Rosa , Bruno Barbara , Pollichieni Simona , Sacchi Nicoletta , Sormani Maria Pia , Fanin Renato , Bandini Giuseppe , Bosi Alberto , Rambaldi Alessandro , Alessandrino Paolo Emilio , Falda Michele , Bacigalupo Andrea
Published: October 2019

Background HLA-matching between patient and donor is critical for outcome of haematopoietic stem cell transplantation (HSCT) performed from an unrelated donor, together with other non-HLA factors. A 10/10-matched donor is the most suitable for HSCT, when possible; however, a certain degree of HLA-incompatibility has to be accepted when such a donor is not available.

Aim of the study This is a prospective multicenter GITMO study in which patients were enrolled as 6/6” matched (matched at loci A, B at low resolution level and DRB1 at high resolution) or as “5/6” matched (one low-resolution mismatch (MM) at locus A or B). High resolution typing is now available as well.

Patients. Eligible were patients with a haematological malignancy, aged > 18 years old, activating an unrelated donor search in Italy from January 1999 to June 2006. Patients with chronic myeloid leukaemia were excluded from the analysis. A total of 1069 patient-donor pairs were analyzed; median follow-up for alive pts was 23 months (range: 3–96). According to disease status at HSCT, pts were classified as “early” (de novo acute myeloid or lymphoblastic leukemia in first complete remission, n=241) and “advanced” (all others, n=828) pts. Univariate and multivariate analysis with Cox regression included main non-HLA factors known to be associated with HSCT outcome. No differences in overall survival (OS) was observed between 5/6-matched (n=101) and 6/6-matched (n=968) pts (fig.1). There was a non-statistically significant trend toward a lower relapse-related death (RRD) in 5/6-matched compared to 6/6-matched pts was observed (5y-RRD: 18% vs 24%, respectively; p=0.13). We then proceeded to test the effect of the C locus: in multivariate analysis, the presence of an antigenic MM at locus C was associated with a worse OS, regardless of matching at loci A or B (RR 1.18, 95% C.I.: 1.00–1.40, p=0.05); the presence of a MM at locus B and/or C significantly affected transplant-related mortality (TRM) (RR 1.57, 95% C.I.: 1.02–2.44, p=0.04; RR 1.31, 95% C.I.: 1.04–1.65, p=0.02, respectively), MM at locus B or C was also associated with development of grade II-IV acute graft-versus-host disease (aGVHD) on multivariate analysis, together with “non-use” of ATG (ATG use: RR 0.70, 95% C.I. 0.52–0.95, p=0.02).

Conclusions, The outcome of so called 5/6 and 6/6 HLA matched unrelated donor transplants is comparable: this may be due to the fact that the C locus is not taken in to account in the “5/6 or 6/6” definition. Indeed a mismatch at locus C is associated with worse outcome. MM at locus B or C, but not at locus A, increases the risk of developing grade II-IV aGVHD. Use of ATG reduces aGVHD rates, thus attenuating the adverse effect of MMs at loci B and C on HSCT outcome.
c-Abl Kinase Inhibitors Overcome CD40-Mediated Drug Resistance in CLL.
Authors: Hallaert Delfine Y. , Jaspers Annelieke , van Noesel Carel J. , Van Oers Marinus H.J. , Kater Arnon P. , Eldering Eric
Published: October 2019

In chronic lymphocytic leukemia (CLL), proliferation centers reside in lymph node (LN) and possibly also bone marrow, where the environment protects CLL cells from apoptotic and cytotoxic triggers. This protective milieu may well contribute to the lack of curative chemotherapy in CLL, and our recent analysis of the distinct profiles of apoptosis regulators in CLL LN versus peripheral blood supports this notion (Smit et al., Blood 2007, 109: 1660). The aim of the present study was to define the molecular basis for the increased drug resistance and to search for novel strategies to circumvent it. To mimic the situation in CLL LN, we applied prolonged in vitro CD40 stimulation of CLL cells, which results in strong upregulation of anti-apoptotic Bcl-xL and Mcl-1. Moreover, we now also report a gradual reduction of Bim at the protein level, further contributing to the anti-apoptotic profile. Using specific inhibitors (PD98059 and MG132), we found that the decrease in Bim is due to ERK-mediated phosporylation and subsequent proteasomal degradation. ERK inhibition during CD40 triggering abrogated the decrease in Bim levels, but did however not re-establish sensitivity to various drugs (fludarabine, Velcade, Roscovitine). In chronic myeloid leukemia (CML), changes in Bcl-xL, Mcl-1 and Bim levels similar to those observed in our CLL/CD40 system are known to depend on BCR-Abl signaling. Therefore, we next applied c-Abl inhibitors Gleevec or Dasatinib in conjunction with CD40. Both drugs caused a profound reversal of most protective CD40 effects; ERK activity, Bim, Bcl-xL and Mcl-1 levels as well as sensitivity to subsequent drug treatment were restored to pre-CD40 values. These effects also occurred in CLL samples with dysfunctional p53 (n=3). Importantly, in CLL LN samples we also found strong ERK activation together with high Bcl-xL and Mcl-1 but low Bim levels, suggesting that there might be a c-Abl dependent survival pathway in proliferation centers. These data provide a molecular basis for combination strategies that could target refractory niches in CLL, using therapeutics that function independently of p53.
The Prognostic Importance of T and NK-Cells in a Consecutive Series of Newly Diagnosed Patients with Chronic Lymphocytic Leukemia.
Authors: Palmer Shanique , Hanson Curtis A. , Zent Clive S. , Porrata Louis F. , LaPlant Betsy , Geyer Susan M. , Markovic Svetomir N. , Call Timothy G. , Bowen Deborah A. , Jelinek Diane F. , Kay Neil E. , Shanafelt Tait D.
Published: October 2019

Background: Patients with CLL have a variable clinical course. Current novel prognostic indicators (ZAP-70, CD38, IgVH gene mutation status, FISH) are able to identify early-stage CLL patients at high risk of rapid disease progression. These prognostic factors focus on characteristics of the malignant B cell clone and are not currently changeable factors. Identification of modifiable characteristics of the host unrelated to the leukemic B-cell but that relate to CLL-specific survival may provide opportunities for therapeutic intervention. Since the immune system is likely to modulate disease progression in CLL patients, we evaluated critical features of that system in relation to the size of the circulating clonal B-cell population in a CLL cohort.

Methods: 186 consecutive patients with CLL who were evaluated at Mayo Clinic within 2 months of diagnosis between 2000 and 2002 were identified. The primary end-point of the study was to assess if host immunity affected time to treatment (TTT). Baseline flow cytometry analysis was available for 166 patients (88%) and was used to calculate the absolute number of T-cell and natural kill (NK)-cells present at diagnosis. The size of the T-cell/NK-cell compartment relative to the size of the malignant monoclonal B-cell (MBC) compartment was then evaluated by calculating the NK:MBC ratio and T:MBC ratio. Relationships with other prognostic parameters and with TTT were evaluated.

Results: Patients exhibited substantial variation in the absolute number of T-cells and NK cells as well as T:MBC and NK:MBC ratios at the time of diagnosis. Higher T:MBC and NK:MBC ratios were observed among patients with early Rai stage and mutated IgVH genes (all P≤0.0002). As continuous variables, both the T:MBC ratio (p value=0.03) and NK:MBC ratio (p value=0.02) were associated with TTT. Since they correlated with TTT as continuous variables, thresholds to classify MBC, T:MBC ratio, and NK:MBC were identified using the method of Contal and O’Quigly. On multivariate Cox modeling including stage, CD38, absolute MBC count, T:MB ratio, and NK:MB ratio, the independent predictors of TTT were disease stage, T:MB ratio, and NK:MB ratio (Table 1).

Conclusion: Measurable characteristics of the host immune system appear to relate to the rate of disease progression in patients with newly diagnosed CLL. These characteristics can be modified and continued evaluation of immunomodulatory drugs, vaccination strategies, and cellular therapies to delay/prevent disease progression are warranted. Additional studies exploring how interactions between the host immune system and the leukemic clone influence clinical outcomes are needed.