bluuebunny/crossref_metadata_2025 huggingface dataset:
Sideroblastic Changes Can Be Recognized in the Erythrogram.
Authors: Rovó Alicia , Passweg Jakob R. , Favre Geneviève , Heim Dominik , Meyer-Monard Sandrine , Tsakiris Dimitrios , Gratwohl Alois , Tichelli André
Published: April 2020

The scatter plots of red blood cells (RBC), called erythrogram produced by the ADVIA® 2120 cell counter gives a visual representation of RBC characteristics. Using a pair of threshold gates on each axis, nine areas are defined according to the cell volume and the hemoglobin (Hb) content. Normal RBC are distributed in the central quadrant. In diseases such as iron deficiency and thalassemia the erythrogram shows a characteristic pattern commonly used for diagnostic approach. We observed in patients, mainly with myeloid diseases with ringed sideroblasts (RS) in the bone marrow a particular erythrogram pattern with a broad distribution of the RBC and a marked variation in RBC size and hemoglobinization. From the central quadrant an abnormal RBC population shifts on an imaginary axis to the lower-left quadrant representing a tail advancing into the microcytic and hypochromic quadrants (Fig1). To confirm whether this particular erythrogram was predictive for bone marrow sideroblastic changes; we compared retrospectively the erythrogram of patients with RS to a group of myeloid diseases without RS. Between January 2004 and August 2005, 33 of 1973 cases with more than 15% of RS in the marrow were identified, 21/33 patients were evaluable (AML=2, MDS=13, MPS=1, MDS/MPS=3, non neoplasia=2). These patients were compared to 30 consecutive cases with myeloid diseases without RS (No RS) (AML=16, MDS=11, MPS=2, MDS/MPS=1). In addition to the erythrogram pattern, Hb, RBC indices, and bone marrow iron staining were analyzed in both groups. We defined two types of erythrogram pattern in respect of sideroblastic changes: a) typical (fig1) b) non typical: any other pattern (i.e. macrocytosis, fig 2). The erythrogram was typical in 17/21 patients with RS and in 0/30 patients with No RS (p<0.0001). The positive predictive value for sideroblastic changes was 100% and the negative predictive value was 88%. Despite the RBC indices comparison showed statistical significance in some variables, they were not specific enough to identify sideroblastic changes. In the group with RS, mean cell Hb was lower (median 30.8 versus 33.6pg), RBC distribution width was higher (19.3 versus 16.5%), the % of hypochromic RBC was higher (5.3 versus 0.9%) and Hb content of reticulocyte was lower (33 versus 37 pg) compared to No RS patients (p<0.05). This last index was particularly useful to rule out iron deficiency in RS group as a cause of hypochromic RBC changes, since in contrast to iron deficiency it was not decreased. In conclusion: sideroblastic changes can be recognized in the erythrogram. Indeed, despite myeloid diseases with RS are a heterogeneous group of diseases they have a common pattern of RBC distribution that can be considered as a kind of fingerprinting for sideroblastic changes with a high predictive value allowing a straightforward diagnostic approach in clinical practice.

Figure 1 Figure 1. Figure 2 Figure 2.
MtDNA Sequence of MDS Patients Treated with CoQ10.
Authors: Sechman Eric V. , Galili Naomi , Einstein Eric , Raza Batool , Ferrante Kimberly , Raza Azra
Published: April 2020

We conducted a pilot study to test the efficacy of coQ10 in improving the cytopenias of MDS patients with low to intermediate risk MDS. Almost every patient experienced a reduction in fatigue and improvement in life quality. Among 25 evaluable patients, 7 responded according to the International Working Group (IWG) criteria. Responses included two trilineage and 1 monolineage response, two cytogenetic responses, and two patients showed a change in FAB type from CMMoL to RA and RARS. CoQ10 is an important coenzyme that has been shown to be involved in protection against oxidative stress, inhibition of apoptosis, and generation of ATP. Mutations in mitochondrial DNA have previously been found in MDS patients. We hypothesized that acquired mutations in the mitochondrial genome may relate to the efficacy of coQ10 treatment. We therefore sequenced the mitochondrial genome (mtDNA) of 9 patients, including 4 responders and 5 nonresponders, and 2 normal samples to determine if differences in the frequency or location of mtDNA mutations could be correlated to response. Whole genomic DNA was isolated from bone marrow monocytes and the mtDNA was PCR amplified in 40 overlapping segments. The PCR products were then sequenced and compared to a mitochondrial genome database (www.mitomap.org) to identify mutations. This technique will not detect mutations present in only a small subset of DNA copies. However, mutations present in a majority of mtDNA that may directly contribute to the expansion of the MDS clone will be identified. Interestingly, although mutations were observed, no differences were found that distinguished responders from nonresponders. We conclude from this analysis that the clinical benefit to MDS patients is not related to specific mutations in the mitochondrial genome. Clinical response may instead be related to other pleiotropic effects of coQ10, such as inhibition of apoptosis.
Research of Subset and Function of Th Cells in Bone Marrow of Myelodysplastic Syndrome Patients.
Authors: Wang Xiuli , Wu De Pei , He Guangsheng , Miao Miao , Sun Aining
Published: April 2020

Objective

To study the quantity and ratio of Th1, Th2 cells in the bone marrow of patients with MDS;The tumor burden that Th1 cells bear in the patients with MDS. Evaluation of the correlation between the ratio of the blast cells and the number of the Th1 cells in the bone marrow in the patients with MDS.

Methods

By FACS, the quantity and ratio of IFN-γ producing CD4+ T cell (Th1) and IL-4 producing CD4+ T cell (Th2) cells in the bone marrow were detected in 21 patients with MDS,18 normal controls and 13 patients with SAA respectively;The karyotypes of 18 patients with MDS and 15 normal controls were detected, The burden,(the number of the cells with anormal karyotypes /the number of all the detected cells) that Th1 cells bear in the patients with MDS and that in normal controls were analyzed;The correlation between the ratio of the blast cells in the bone marrow and the number of the Th1 cells in the patients with MDS was also analyzed.

Results

The percentages of Th1 cells and Th2 cells and ratio of Th1/Th2 in the bone marrow of normal controls were: (0.48±0.10)%,(0.24±0.19)%,2.31±0.76 respectively, while those of the patients with MDS were (0.36±0.11)%,(0.76±0.35)% and 0.51±0.13. The percentage of Th1 cells of patients with MDS was reduced and the ratio of Th1/Th2 of them was significantly lower than that of normal controls(p<0.01); Those of the patients with SAA were: (4.75±0.49)%,(0.40±0.28)%,26.5±8.79 respectively, their Th1 cells and ratio of Th1/Th2 were markedly higher than those of normal controls (P<0.01); In all of the 15 normal controls the karyotypes were normal, and tumor cell burden was zero, While in the group of MDS, tumor cell burden, (the number of the cells with anormal karyotype /the number of all detected cells) was (50.00±0.10)%. The MDS patients’ ratio of tumor cell burden / Th1 cells (1.72±1.23) was much higher than that of normal controls whose burden was zero;The lower ratio of the Th1 cells in the bone marrow of the patients with MDS and the AML which converts from MDS, the higher percentage of the blast cells, they were negatively correlated (r=−0.563, p<0.01).

Conclusion

The immune function of T lymphocytes in MDS is abnormal: the balance between Th1 and Th2 cells is broken. Th1 cell, the most important element for antitumorigenesis, is decreased. Comparing with the burden of the malignant clone, the number of Th1 cells of the patients with MDS is overwhelmingly scarce. With descending of the number of Th1 cells in the bone marrow of the patients with MDS, the number of the blast cells contrarily grows. MDS is a heterogeneous group of neoplastic disease accompanied with hypofunction of the T lymphocytes mediating immune surveillance.

All these hint that during the treat of clearing the malignant clone of MDS, enhancement of the T cell fuction should been done.
Usefulness of Functional Image Analysis Using Positron Emission Tomography for the Discrimination between Hypoplastic Myelodysplastic Syndromes and Aplastic Anemia.
Authors: Takatoku Masaaki , Sato Toshihiko , Hatano Kaoru , Omine Ken , Mori Masaki , Muroi Kazuo , Nagai Tadashi , Ozawa Keiya
Published: April 2020

Myelodysplastic syndromes (MDS) and aplastic anemia (AA) are classified bone marrow failure syndromes. Their clinical and pathological features partly overlap. It is sometimes difficult to distinguish hypoplastic MDS from AA by using current common diagnostic methods such as bone marrow biopsy and chromosome analysis. Although magnetic resonance image (MRI) is useful for diagnosis of MDS with hypercellular bone marrow, it is difficult to discriminate between hypoplastic MDS and AA using this method because the high intensity patterns on T1-enhanced images are similar in these disorders. Quantitative imaging with fluorine-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) has been recognized as a useful examination for discrimination between benign and malignant regions in various conditions. Since the decrease in FDG uptake at the late phase is much slower in a malignant region than in a benign region, dual time point imaging provides more accurate information than does conventional single time point scanning. In this study, we investigated the usefulness of dual time protocol FDG-PET in differential diagnosis of hypoplastic MDS and AA. Seventeen patients (7 with typical MDS (RA), 4 with hypoplastic MDS, 6 with AA) and 30 healthy adults agreed to participate in this study. Bone marrow biopsy, FDG-PET, MRI and CT were carried out. Spine, femur and iliac lesions were detected by their increased FDG uptake at 60 and 120 min after injection of 0.12 mCi/kg of FDG. The mean lesional standardized uptake values (SUVmean) at 60 and 120 min after injection of FDG were determined. While the median SUVmean of normal lumbar regions at 60 min was 1.123 ± 0.219, that of MDS cases was 2.06 (range, 1.62–2.32). At 120 min, the median SUVmean of normal cases was 1.180 ± 0.119, whereas that of MDS cases was 2.44 (range, 2.08–2.98). It is noteworthy that the SUVmean of four hypoplastic MDS cases was also high (ranges: 1.96–2.23 at 60 min and 1.97–2.52 at 120 min), suggesting that bone marrow in hypoplastic MDS has a hyper-metabolic state of glucose like other malignant disorders. We also observed a patchy hot area, which may be a visualization of ineffective hematopoiesis, throughout the spine image of hypoplastic MDS. In contrast, the SUVmeans at both time points of four AA cases were not different from those in normal control cases. These results suggest that functional imaging analysis using an FDG-PET dual time protocol enables discrimination between hypoplastic MDS and AA, in which MR images similar intensities.
Detection of sjTRECs within Peripheral Blood T Cells Evaluation of Recent Thymic Output Function in Myelodysplastic Syndrome.
Authors: Du Xin , Li Yang-qiu , Weng Jian-yu , Lu Ze-sheng , Guo Rong , Wu Sui-jing , Zhong Li-ye , She Miao-rong , Lin Wei , Huang Zi-lun
Published: April 2020

Objective

The myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem cell disorders, while, immunological abnormalities are frequently observed in patients with MDS. Several reports revealed that about 10% of MDS patients have clinical autoimmune disorders like skin vasculitis, rheumatic disease, or autoimmune hemolytic anemia. Furthermore, serological immunological abnormalities like hyper- or hypogammaglobulinemia, positivities of antinuclear antibody, positivities of direct Coombs test, or inverted CD4/8 ratios were found in 18–65% of patients with MDS. Recently immunosuppressive therapies including prednisolone, antithymocyte globulin, and cyclosporin A (CsA) are used to treat cytopenia in some patients with MDS. But it isn’t very clear the immunosuppressive mechanism in MDS and the value of the treatment. To analyze the content of signal joint Tcell receptor excision DNA circles signal joint T cell receptor excision DNA circles(sjTRECs) within peripheral blood mononuclear cells (PBMCs), thereby to infer the level of naive T cells and the recent thymic output function in patients with myelodyspoastic syndrom. Methods Quantitative detection of sjTRECs in DNA of peripheral blood mononuclear cells from 13 normal individuals and 8 patienets were performed by real-time polymerase chain reaction (PCR) and TaqMan technique.

Results

The median value of sjTRECs copies P1 000 PBMCs was 4.37±3.64 in normal individuals whereas it was1.07 ±1.40 copies P1 000 PBMCs in myelodysplastic syndrom patients (P <0. 05). Conclusions MDS Patients decrease in recent thymic output function.
Analysis of the T-Cell Receptor Repertoire in the Study of Immunological Abnormalities in Myelodysplastic Syndromes (MDS).
Authors: Du Xin , Li Yangqiu , Weng Jianyu , Zhong Liye , Chen Shaohua , Yang Lijian , Lu Zesheng
Published: April 2020

Introduction

The myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem cell disorders, while, immunological abnormalities are frequently observed in patients with MDS[1]. Several reports [2,3]revealed that about 10% of MDS patients have clinical autoimmune disorders like skin vasculitis, rheumatic disease, or autoimmune hemolytic anemia. Furthermore, serological immunological abnormalities like hyper- or hypogammaglobulinemia, positivities of antinuclear antibody, positivities of direct Coombs test, or inverted CD4/8 ratios were found in 18–65% of patients with MDS. Recently immunosuppressive therapies including prednisolone, antithymocyte globulin, and cyclosporin A (CsA) are used to treat cytopenia in some patients with MDS. We reported four patients with MDS. Rearrangements of the TCR-beta genes were seen in these patients using RT-PCR and Genescan analysis (CDR 3 length analysis). Also they had skewed TCR usages using TCR repertoire analysis.

Methods

Two patients with refractory anemia(RA), two with refractory anemia with blasts(RAEB). four males from 41to 68 yearsold. Complementarity determining region 3(CDR3) of TCR Vβ with 24 variable region gene was amplified in peripheral blood mononuclear cells, which were drawn from five patients with myelodysplastic syndromes (MDS) using RT-PCR, to observe the expression of TCR Vβreceptoroire T cells, the PCR products were further analyzed by genescan to evaluating clonality of T cells (CDR 3 length analysis), and compare results with age-matched healthy donors and patients with graft versus host disease(GVHD).

Results

We found a significantly higher number of skewed Vb profiles in the MDS and GVHD patients compared with donors. In peripheral blood T cells, Only 2-11 Vb subfamily T cells could be identified in MDS patinets, clonal expansion T cells could be found in Vb1, 3,13, 14 and 21 subfamilies.

Disussion

In this study, we evaluated the total T-cell repertoire of 4 MDS patients using gendscan analysis to look for evidence of T-lymphocyte clonality. This analysis showed that all 4 patients exhibited extensive skewing of their TCR spectratypes, suggesting clonal or oligoclonal T-cell expansions. Epperson [10]also reported the same results. As we know, acute and chronic graft-versus-host disease(GVHD) is a major complication after allogeneic bone marrow transplantation. GVHD is mediated by T cells that are derived from the BM graft. In this study, we compared the results of GVHD patients with that of MDS patients, and found that these two groups patients show TCR Vb skew distribution and clonal expansion. These findings provide further evidence that T cell mediated immune processes are a feature of MDS.
Anti-Tumor Activity of a Novel Bcr-Abl/Lyn Dual Inhibitor, NS-187, in Murine CNS Ph+ Leukemia Models.
Authors: Yokota Asumi , Kimura Shinya , Oyama Tatsuya , Ashihara Eishi , Naito Haruna , Urasaki Yoshimasa , Ueda Takanori , Terui Yasuhito , Hatake Kiyohiko , Hirabayashi Kazuko , Maekawa Taira
Published: April 2020

The penetration of imatinib mesylate (Gleevec™) into the central nervous system (CNS) is poor. Hence the CNS becomes a sanctuary site for patients who are on prolonged imatinib therapy. P-glycoprotein (P-gp) plays an important role in limiting the distribution of imatinib to the CNS, and it is well known that imatinib is a substrate of P-gp. We have recently identified a specific dual Bcr-Abl/Lyn inhibitor, NS-187, which can override imatinib-resistance. NS-187 was 25–55 and at least 10 times more potent than imatinib in vitro and in vivo, respectively. The purpose of this study was to investigate whether NS-187 can inhibit the growth of Ph+ leukemic cells in the CNS. In our preliminary pharmacokinetic study, the intracranial concentration of NS-187 was 10% of its serum concentration, suggesting the involvement in P-gp. To determine whether NS-187 is effluxed by P-gp, we examined the growth-inhibitory effects of NS-187 alone and in combination with a P-gp inhibitor, verapamil or cyclosporin A, on K562 cells and on a multidrug-resistant (MDR) K562/D1-9 cell line overexpressing P-gp. The K562/D1-9 cell line was 10 times more resistant to NS-187 than the parental K562 cell line, and P-gp inhibitors abolished this resistance, indicating that the action of NS-187, like that of imatinib, is affected by the P-gp-related MDR system. Even though NS-187 was found to be a substrate for P-gp, it inhibited the growth of K562/D1-9 cells at a concentration which could be achieved in the brain. we therefore tested the anti-tumor effects of NS-187 in murine CNS leukemia models. mice were inoculated into right cerebral ventricle with 1×105 BaF3/wt bcr-ablGFP cells (Balb/c-nu/nu mice) or 1×106 K562GFP cells (NOD/SCID mice). Five days after inoculation, mice were randomized into groups of 4 and orally administrated twice a day with vehicle, imatinib or NS-187 for 14 consecutive days. Sixteen days after inoculation, three mice from each group were sacrificed and their brains were examined under a fluorescent stereoscopic microscope. NS-187 inhibited the proliferation of leukemic cells in the brain, whereas imatinib did not. Moreover, NS-187 significantly prolonged the survival of the mice in a dose-dependent manner in both murine models compared with imatinib (Figure). In conclusion, NS-187 can inhibit Ph+ leukemic cell growth in the CNS in spite of efflux of the compound by P-gp.

Figure Figure
Inherited Thrombophilias in Essential Thrombocythemia and Polycythemia Vera Are Not an Additional Risk Factor for Thrombosis.
Authors: Cacciola Emma , Cipolla Antonio , Di Francesco Ernesto , Giustolisi Rosario , Cacciola Rossella R.
Published: April 2020

Patients with essential thrombocythemia (ET) and polycythemia vera (PV) have a high risk of thrombosis. Inherited thrombophilias are established risk factors for thrombosis. To determine if inherited thrombophilic abnormalities might contribute to the thrombotic risk in ET and PV, we investigated antitrombin III (ATIII), protein C (PC) and protein S (PS), polymorphisms of clotting factors V (FVQ506) and II (G20210A) and methylenetetrahydrofolate reductase (MTHFR) mutation in patients with ET and PV and we tried to correlate these results with prothrombotic markers such as bthromboglobulin (bTG), platelet factor 4 (PF4), prothrombin fragment 1+2 (F1+2) and d-dimer (DD). In addition, we analysed a possible association between inherited coagulopathy and thrombosis. ATIII, PC and PS antigen levels were assayed by ELISA as well as bTG, PF4 and F1+2. PCR-based assays were used for FVQ506, prothrombin and MTHFR. DD was measured by immunoturbimetric latex agglutination. The study involved 101 patients diagnosed with ET (71 patients, 30 men, 41 women, mean age 62.3 years) or PV (30 patients, 21 men, 9 women, mean age 65.4 years) according to conventional criteria. The duration of disease ranged between 1 to 12 years. Of 101 patients, 53 (52,47%) were non-carriers of mutations and among these 24 (45.28%) experienced thrombosis, 48 (47.52%) were carriers and 27 (56.25%) had thrombosis. With reference to carriers, 9/48 (14.58%) had combined mutations such as PC and PS (2/9), PS and G20210A (1/9), PC and MTHFR (4/9), G20210A and MTHFR (2/9). The prevalence of studied defects was 31.25% (15/48) and 18.75% (9/48) for deficiency of PC and PS, respectively, 8.33% (4/48) and 14.58% (7/48) for heterozygosity of FVQ502 and G20210A, respectively, and 45.83% (22/48) for MTHFR mutation. Deficiency of ATIII was not present. All patients had higher bTG, PF4 and F1+2 (365±654 IU/ml, 133±64 IU/ml and 2.6±2.5 nmol/L, respectively) than to controls (23.8±9.1 IU/ml, 5.5±2.8 IU/ml and 0.60.2 nmol/L, respectively) (p<0.0001) and normal DD (144±92 ng/L). No correlation between studied polymorphisms genetic and bTG, PF4, F1+2 and DD was found as well as with thrombotic complication. The frequency of thrombosis was not statistically different between non-carriers and carriers. Based on these data, it appears that inherited thrombophilias are not an additional risk factor for thrombosis in ET and PV and that the thrombotic phenomena are due to the abnormal platelet function that is part of the myeloproliferative disorders.
Identification of Bcr/Abl Point Mutations Conferring Resistance to the Abl Kinase Inhibitor AMN107 by a Random Mutagenesis Study.
Authors: Ray Arghya , Cowan-Jacob Sandra , Manley Paul W. , Mestan Jurgen , Griffin James D.
Published: April 2020

The development of kinase inhibitors such as imatinib that block the Bcr/Abl tyrosine kinase has significantly improved chronic myeloid leukemia (CML) therapy. However, patients with advanced disease often develop resistance to imatinib due to the emergence of clones with point mutations in the tyrosine kinase domain. AMN107, a novel second generation inhibitor of Bcr/Abl (Weisberg et al., Cancer Cell, 7:129, 2005) is currently in Phase 2 clinical trials and shows significant clinical activity in some patients with imatinib-resistant CML. However, it is possible that resistance to AMN107 could occur through the emergence of new Bcr/Abl point mutations, and here we report the results of a random mutagenesis study to identify Bcr/Abl mutants selected for resistance to AMN107. A library of mutations was generated in the target gene by propagating a native BCR-ABL-GFP retroviral construct through a bacterial strain deficient in a DNA repair pathway. Murine Ba/F3 cells were then transfected/infected with the mutated vector and subsequently the cells were selected for the ability to proliferate in the presence of AMN107 (0.125–0.5 μM). The Ba/F3 cells expressing native Bcr-Abl did not grow under these conditions. Single cell clones were expanded and a total of 60 individual colonies were isolated for which BCR/ABL was sequenced. Twenty colonies had single point mutations located in the kinase domain of the BCR-ABL gene. The rest had multiple point mutations and were not considered for further analysis. The point mutants identified in this way were all validated by preparing the corresponding Bcr-Abl cDNA using site-directed mutagenesis, generating a new mutant-Bcr-Abl/Ba/F3 cell line and testing for resistance to both AMN107 and imatinib. The mutant cell lines confer varying degrees of resistance to AMN107, from 5- to 400-fold. The mutant variants identified in this study included 15 novel mutations and 5 known imatinib-resistant mutations that have previously been identified in CML patients. These latter mutations included, T315I, which similar to imatinib, showed maximum resistance against AMN107 (~50% survival at 10 μM concentration). Interestingly, the majority of novel AMN107-resitant mutants were also found to be resistant to imatinib and the rank order was highly correlated to the rank order of resistance to AMN107. These data may be helpful in providing insights into the mechanism of acquired resistance of Bcr-Abl to small molecule inhibitors and are likely to predict some of the resistance mutations that may be observed in the clinic.
Myeloproliferative Disease in a Mouse Model of Down Syndrome.
Authors: Mundschau Gina , Jilani Sarah , Liu Hui , Davis Elizabeth , Le Beau Michelle , Crispino John
Published: April 2020

Trisomy 21 causes human Down syndrome (DS), a heterogeneous group of phenotypes including marked predisposition to leukemia. Children with Down syndrome are 500 times more likely to develop acute megakaryoblastic leukemia (AMKL) than other children. Furthermore, non-DS children with AMKL often have acquired trisomy 21 in their leukemic clones, suggesting that trisomy 21 in hematopoietic cells contributes to leukemic transformation. To better understand the impact of trisomy 21 on blood cell homeostasis and leukemia, we studied hematopoiesis in the Ts65Dn mouse model of Down syndrome. Ts65Dn mice harbor a segmental trisomy for mouse chromosome 16, homologous to human chromosome 21, and display many of the phenotypes associated with human DS, including craniofacial anomalies and learning deficits. To define the hematopoietic parameters for this strain, we performed monthly complete blood counts for a cohort of trisomic mice and their disomic littermates and discovered the development of progressive thrombocytosis and mild anemia in trisomic animals. Increased numbers of CD41+ megakaryocytes with lower modal ploidy were detected in the bone marrow and spleen of Ts65Dn mice as early as three months of age. Over time, expansion of the megakaryocyte population was accompanied by a decrease in TER119+ cells in the bone marrow, myelofibrosis, splenomegaly, and extramedullary hematopoiesis. Colony forming assays demonstrated increased colony forming ability in the spleens of trisomic mice along with variable decreased hematopoiesis in the bone marrow. Further, characterization of stem cells in the bone marrow indicated a hyperproliferative stem cell population. Importantly, the mice did not develop malignant leukemia by the age of 18 months and no mutations were found in the blood transcription factor GATA1, which is commonly affected in human AMKL. While Ts65Dn mice do not develop the AMKL seen in humans with DS, our results indicate that, trisomy 16 can cause hyperproliferation of the myeloid lineages, extramedullary hematopoiesis, and bone marrow fibrosis in mice. Additionally, these results suggest that trisomy 21 in humans may initiate a similar process in hematopoietic stem cells, which may contribute to leukemogenesis. Unexpectedly, this phenotype also bears significant resemblance to the human myeloproliferative disease chronic idiopathic myelofibrosis (CIMF). These findings may provide insight into the origins and progression of human myeloid diseases, including AMKL and CIMF.
Decitabine: Where Is the Target?.
Authors: Tamm Ingo , Sattler Nicole , Wagner Mandy , Lübbert Michael , leCoutre Philipp , Dörken Bernd , Schmelz Karin
Published: April 2020

The methylation inhibitor 5-Aza-2′-deoxycytidine (decitabine) has therapeutic efficacy in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Using microarray analysis, we investigated global changes in gene expression after decitabine treatment in AML. In the AML cell line OCI-AML2, decitabine induced the expression of 81 out of 22 000 genes; 96 genes were downregulated (X2-fold change in expression). RT-PCR analysis of 10 randomly selected genes confirmed the changes of expression in AML cells. Similar results were obtained with primary AML and MDS cells after treatment with decitabine ex vivo and in vivo, respectively. In contrast, significantly fewer changes in gene expression and cytotoxicity were detected in normal peripheral blood mononuclear and bone marrow cells or transformed epithelial cells treated with decitabine. Interestingly, only 50.6% of the induced genes contain putative CpG islands in the 5′ region. To further investigate the significance of promoter methylation in the induced genes, we analyzed the actual methylation status of randomly selected decitabine-inducible genes. We detected hypermethylation exclusively in the 5′ region of the myeloperoxidase (MPO) gene. DNA methylation inversely correlated with MPO expression in newly diagnosed untreated AML patients (P<0.004). In contrast, all other analyzed decitabine-inducible genes revealed no CpG methylation in the promoter region, suggesting a methylation-independent effect of decitabine.
The Use of the PFA-100® and Thromboelastograph in Assessing Haemostasis and Drug Efficacy in Essential Thrombocythemia.
Authors: Cheung Betty Y.Y. , Parmar Kiran , Yadegarfar Ghasem , Hunt Beverley J. , Harrison Claire N.
Published: April 2020

Introduction. Essential thrombocythemia (ET) is associated with an increased risk of thrombosis. Many patients are treated with anti-platelet agents and/or cytoreductive therapy in order to minimise the risk but some still develop thrombotic events. The PFA-100® is an effective near-patient platelet function analyser and the Thromboelastograph (TEG®) Haemostasis Analyser assesses global haemostasis in whole blood. The usefulness of these technologies has not been fully evaluated in ET.

Patients and Methods. We compared results from the PFA-100® and TEG® in 25 ET patients and 19 controls. Overall the ET patients comprised 18 females and 7 males, their median age was 55 yrs, median platelet count 423x109/l. Twenty-two were on anti-platelet therapy (21 on 75mg aspirin, 1 on aspirin and clopidogrel). Seventeen patients were receiving cytoreductive therapy (10 hydroxyurea, 2 a-interferon, 4 busulphan and 1 busulphan with anagrelide). Eight patients had previous thrombosis (4 transient ischaemic attacks, 1 stroke, 1 central retinal vein occlusion, 2 multiple events [1 stroke, erythromelalgia and digital ischaemia; 1 central retinal vein occlusion and digital ischaemia]) and 2 had haemorrhagic events.

Results. All had normal INR, APTT and fibrinogen levels. Using the PFA-100®, Collagen/ADP closure times were prolonged in 15 (60%) patients compared to normal controls, suggesting platelet dysfunction, which included both patients with previous bleeding episodes. Twenty (91%) patients on aspirin had prolonged Collagen/Epinephrine closure times but for 2 (9%) patients these were within the normal range indicating aspirin-resistance. Both these patients had a history of thrombosis whilst on aspirin alone. Using TEG®, ET patients when compared to controls, took longer to initiate and to reach maximal clot strength (significantly longer R and K times, p=0.001, p=0.012 respectively and smaller angle, p=0.01) but the developed clot had a higher tensile strength (larger MA, p=0.024). There was a positive correlation between fibrinogen levels and MA (r=0.54, p=0.006) as well as APTT with R time (r=0.437, p= 0.029). There was no correlation between platelet count or prior history of thrombosis with any PFA-100® or TEG® variables.

Conclusion. Low-dose aspirin had biochemical efficacy as assessed by PFA-100® Collagen/Epinephrine closure times in most but not all ET patients. Further studies are necessary to assess whether Collagen/ADP times can identify those at risk of bleeding. In ET, the TEG® demonstrates slower formation but a higher tensile clot strength than controls. Larger studies are needed to fully evaluate these findings and their clinical relevance.
Follow-Up of Clinical Manifestations and Platelet Function Test in Patients with Essential Thrombocythemia on Anagrelide Treatment.
Authors: Vassallu Patricia S. , Goette Nora P. , Glembotsky Ana C. , Heller Paula G. , Kornblihtt Laura I. , Lev Paola R. , Chazarreta Daniel C. , Salim Juan P. , Laguna Maria S. , Marta Rosana F. , Molinas Felisa C.
Published: April 2020

Introduccion: Patients with essential thrombocythemia (ET) may have abnormalities of platelet function (PF), the most frecuently are reduced response to epinephrine and ADP-induced aggregation and spontaneous aggregation. Anagrelide (A) has a phosphodiesterase inhibitor activity and may inhibit PF. Therefore, we evaluated the follow-up of clinical manifestations (CM) and the PF in a group of 42 ET patients before treatment and on hematological remission.

Materials and methods: 42 ET patients (33/9, F/M) who meet de PVSG criteria, were studied before treatment with A and on hematological remission. The CM were grouped into thrombosis (T), hemorrhages (H), microcirculation disturbances (MD) and combined (C), that included H plus MD or H plus T. Platelet function tests were perfomed with different agonists such as epinephrine, ADP, ristocetine, arachidonic acid and bovine vWF.

Results: are shown in Table I.

Abbreviations: T≠ (4 TIAs, 1 stroke, 1 AMI), H* (1 upper gastrointestinal bleeding, 4 minor hemorrhages) C± (1 mesenteric, portal and splenic vein thrombosis plus minor hemorrhage, 4 minor hemorrhages plus MCD), Tψ (1 TIA, 1 stroke), H§ (1 lower gastrointestinal bleeding) C† (1 AIT plus minor hemorrhage).

During hematological remission spontaneous platelet aggregation disappeared in all patients except one. Abnormal epinephrine response desappeared in 16 patients, was not corrected in 10 and this abnormality appeared in 2 whose response were previously normal. Abnormal ADP response disappeared in 4 cases during treatment and this abnormality was found in 5 previously normal cases. No correlation was found between spontaneous platelet aggregation and clinical manifestations p= NS. No correlation was found between both epinephrine and ADP response and hemorrhages. No significant abnormalities were found in the response to arachidonic acid, ristocetin, and bovine vWF before or during treatment with anagrelide.

Conclusions: In this group of ET patients platelet function was not affected by the phosphodiesterase inhibibitor activity of anagrelide. Similarly bleeding manifestations were not worsen during this therapy. As previously shown spontaneous platelet aggregation desappeared in all but one patient. We have not yet an explanation for the increased microvascular disturbances during treatment.

Table I Pre treatment On treatment Clinical Manifestations (42 patients ) Asymptomatic 16 18 Thrombosis 6≠ 2ψ Hemorrhages 5* 4§ Microvascular disturbances 10 17 Combined 5± 1† Platelet function Spontaneous aggregation 17/42 1/41 Abnormal epinephrine response 26/41 12/42 Abnormal ADP response 17/41 9/41
Function of CD39 (NTPDase-1) in Chronic Lymphocytic Leukemia.
Authors: Pulte E. Dianne , Olson Kim , Islam Naziba , Broekman M. Johan , Ballard Harold , Marcus Aaron J.
Published: April 2020

Chronic lymphocytic leukemia (CLL) is characterized by accumulation of large numbers of mature lymphocytes. However, thrombotic events in CLL are relatively rare. CD39, the main control system for blood fluidity, was identified on the surface of CLL cells in about 80% of patients in two recent studies. This suggests that CD39 could play a role in prevention of thrombotic events in CLL. Lymphocytes were prepared from peripheral blood of an initial 13 patients with CLL by histopaque isolation and purified to at least 95%. The ADPase and ATPase activities in these lymphocytes was measured by our radio-thin-layer chromatographic assay. Additionally, cells were stained with antibodies to CD3, CD19, and CD39, and analyzed by FACS. Remaining lymphocytes were pelleted and frozen for semi-quantitative RT-PCR analysis of the CD39 transcipt. Lymphocytes from patients with CLL showed increased ATPase and ADPase activities compared with lymphocytes from normal donors. Average ADPase activity per 50,000 cells from CLL patients was 57pmol/min, compared with 14pmol/min in lymphocytes from normal donors. Average ATPase activity was 43.6pmol/min in CLL patients, and 10.6 pmol/min in normal lymphocytes. Semi-quantitative RT-PCR of CLL lymphocytes showed upregulation of CD39 RNA compared with normal lymphocytes. FACS data showed that the percentage of cells expressing CD39 was greater in CLL cells (62.5%) than in normal lymphocytes (11%). Additionally, some CLL cells appear to express CD39 at higher densities than normal lymphocytes. Interestingly, one patient in whom lymphocytes were isolated before and during an accelerated phase of the disease showed a decrease in CD39 expression on his lymphocytes as the disease progressed. Two other patients with advanced disease, requiring chemotherapy, showed relatively low CD39 levels as well. We conclude that CD39 is present and functions as an ecto-nucleotidase on the surface of most malignant lymphocytes in CLL. The quantities are much higher than observed in normal lymphocytes. CD39 may be protective from thrombotic events in patients with CLL. In addition, lymphocyte CLL may be a marker of earlier or less aggressive disease and a decrease of CD39 on the cell surface may signify a worsening of the disease.
Toll like Receptors (TLRs) in B Cell Chronic Lymphocytic Leukemia (B-CLL).
Authors: Manoussaka Maria S. , Memon Azka , Nathwani Amit , Porakishvili Nino , Clark Edward , Lydyard Peter M.
Published: April 2020

Introduction: B-CLL is characterised by the accumulation of long-lived CD5+ B cells. Mature B cells can be activated via TLRs to produce immunoglobulin and proliferate. We have previously reported differential surface expression of CD180, TLR-4 associated receptor, by B-CLL cells in relation to IgVh gene status (Porakishvili N et al., 2004; Blood (ASH annual meeting abstracts) 104: 2807). To further characterise subsets of leukemic cells, we assessed B-CLL cells for TLR mRNA expression.

Method: B cells from untreated (n=24) and treated (CHOP n=3, Thalidomide n=1 and chlorambucil and prednisolone n=2) CLL patients, and healthy aged matched controls, (n=14) were isolated by CD19+ selection using iMACs columns (Miltenyi Biotec), total RNA extracted (Qiagen) and primers for TLR2, TLR4, TLR9, CD180 and the adaptor protein MYD88 were used for TaqMan PCR analysis (ABI). GAPDH was used for normalization. Data were expressed as mean delta cycle number (ct) ± standard deviation, giving a value inversely related to the level of expression of the specific gene. Patients were separated into groups according to IgVh gene mutation status or level of Zap 70 expression. Statistical analysis was performed using by Mann-Whitney U test and Spearman rank correlation or Pearson’s correlation coefficient.

Results: Overall, there was a heterogeneous pattern of TLR expression in patients and controls with no significant differences between them. Treated patients were characterised by higher mRNA levels for the all TLRs measured (TLR9 p=0.018) and MYD88 (MYD88 p=0.026) compared with untreated patients, despite no significant difference of their GAPDH levels (p>0.05). Significant correlations were seen between mRNA levels for the TLRs within the age matched control group (all p<0.05). Groups of patients were further analysed according to mutated (mutated >2%) or unmutated IgVh genes (unmutated <2%). No significant correlation in the TLR expression levels was found within the mutated patient group (n=8) or Zap 70 low expression group (<0.7 level) (n=11). Preliminary data from patients with an unmutated IgVh gene status (n=4) or according to levels of ZAP 70high expression (n=5) showed correlation of mRNA levels for TLRs and MYD88 expression (all p<0.05).

Conclusion: Our data indicates a direct relationship between MYD88 and TLRs transcription levels in healthy control individuals. Lack of significant correlation amongst TLR mRNA levels in CLL patients with mutated IgVh genes and Zap70low expression levels suggests aberrant regulation of TLRs in this group.
Characteristics and Outcome of B-Cell Chronic Lymphocytic Leukemia in HCV-Positive Patients.
Authors: Molica Stefano , Mirabelli Rosanna , Misuraca Demetrio , Battaglia Caterina
Published: April 2020

HCV-associated B-cell non-Hodgkin’s lymphomas (NHL) show distinctive clinico-pathological features such as older age, liver damage, presence of monoclonal gammopathy, increased incidence of autoimmune disorders, extranodal localizations and restricted histological subtypes. As far as B-cell chronic lymphocytic leukemia (CLL) is concerned, information dealing with either characteristics or outcome of HCV-associated CLL are limited. With this background we compared clinico-hematological features and outcome of 34 HCV-positive patients diagnosed at our institution as having immunologically typical B-cell CLL (i.e., CD5+/CD23+/CD79b-/SmIg dim) with 161 unselected CLL HCV-negative patients followed-up in the last 10 years. The two groups were alike with respect to main clinico-hematological features such as age (P=0.780), sex (P=0.650), absolute lymphocyte count (P=0.788), platelet count (P=0.362), haemoglobin level (P=0.704), β2-microglobulin (P=0.192), Binet stage distribution (P=0.224) and lymphocyte doubling time (LDT)(P= 0.620). As expected either ALT or AST serum levels at the time of CLL diagnosis were significantly higher in HCV-positive patients in comparison to HCV-negative ones (P<0.0001 for both). In contrast, no difference was found in the incidence of monoclonal gammopathy between HCV-positive and HCV-negative patients (10.3% versus 7.7%; P=0.708). The same applied for autoimmune disorders which were homogeneously distributed in the two subgroups (P=0.711) and accounted, more frequently, for autoimmune emolytic anemia (AEA)(HCV-negative subgroup, 5.5%; HCV-positive subgroup, 9.0%). The proportion of severe infections registered did not reflect the HCV-status (HCV-negative subgroup, 9.6%; HCV-positive subgroup 6.4%; P= 0.510). Also second tumours were equally distributed among HCV-positive and HCV-negative subgroups (10% versus 6.8%; P=0.655). Survival curves projected at 10 years did not show any statistical in terms overall survival (Hazard Risk, 0.690; 95% CI: 0.216–1.304; P=0.167). Finally, the short term hepatic toxicity of chemotherapy did not increase among HCV-positive patients (P=0.671).

In conclusion, HCV-positive patients with B-cell CLL do not differ from other patients both for presentation and clinical outcome. The need to activate specific protocols of antiviral therapy appears less urgent in comparison to NHL, however, younger CLL patients HCV-positive who are eligible for therapies at higher immunosuppressive potential (i.e., chemo-immunotherapy) should be taken in special consideration.
Polymorphisms in the Promoter Region of the Mcl-1 Gene Are Associated with Ex Vivo Cytotoxicity but Not with Response to Treatment in Advanced Chronic Lymphocytic Leukemia (CLL).
Authors: Villamor Neus , Marce Silvia , Bosch Francesc , Ferrer Anna , Pérez-Galán Patricia , López-Guillermo Armando , Giné Eva , Campo Elías , Montserrat Emili , Colomer Dolors
Published: April 2020

High levels of Mcl-1 have been related to a poor response to therapy in patients with CLL. Genetic polymorphisms in the promoter region of the Mcl-1 gene consisting in insertions of 6 (+6) or 18 (+18) bp have been described in 30% to 60% of patients with CLL. The biological and clinical translation of such polymorphisms is controversial. We have analysed this issue in a cohort of newly diagnosed patients with CLL (14 F/ 46 M; 80% in B or C Binet’s stage), treated with a combination of fludarabine (F), cyclophospamide (C) and mitoxantrone (M). Polymorphisms were correlated with:

Mcl-1 RNA expression as assessed by quantitative PCR, ex vivo cytotoxicity against F, C, and M, alone or in combination, using the MTT assay, and clinical response to treatment.

The frequency of the wild type (wt), +6, and +18 allel was 56%, 12%, and 32 %, respectively (table). Twenty-one of 60 (35%) patients were homozygous for wt allel. Quantification of Mcl-1 RNA expression was performed in 18 patients. Wt/wt patients (n=6) tended to have lower Mcl-1 gene expression than the remaining patients (1.0 AU ±0.4 vs 2.2 AU ±2.8). Ex vivo cytotoxicity was different according to the Mcl-1 genotypes for F, M, F+M, and C+F, but not for FCM. The highest cytotoxicity was observed in patients with +18/+18 genotype (n=7) while those with wt/wt (n=11) presented intermediate cytotoxicity and those with other genotypes (n=17) had the lowest cytotoxicity. Twenty-eight patients achieved CR, 22 PR, and 5 failed to respond. No relationship was found between Mcl-1 polymorphisms and response to therapy (table). Interestingly, however, the ex vivo response to F (58.2% ±16 vs 45.7% ±16, p=0.05) and F+M (87% ±9 vs 76% ±15.5, p=0.04) was greater in the 13 patients who achieved MRD-negative CR. After a median follow up of 28 months (range: 6–53), 16 of 38 patients have progressed. No relationship was found between Mcl-1 polymorphism and progression after treatment. In summary, in this series of patients with advanced CLL homogeneously treated the frequency of insertions in the promoter region of Mcl-1 gene was high. Although Mcl-1 polymorphisms were associated with the ex vivo response to antileukemic drugs, they did not correlate with response to treatment and disease progression.

Distribution of Mcl-1 Polymorphisms and Response to Treatment Allel Allelic Frequency Genotype Patients CR (MRD- CR) wt/wt 21 (35%) 9 ( 4 ) wt 68 (56%) +6/+6 2 ( 3%) 0 ( 0 ) +18/+18 7 (12%) 4 ( 3 ) +6 14 (12%) wt/+6 6 (10%) 4 ( 2 ) wt/+18 20 (33%) 9 ( 3 ) +18 38 (32%) +6/+18 4 ( 7%) 2 ( 1 )
Aggressive Cutaneous Squamous Cell Carcinoma (SCC) in Chronic Lymphocytic Leukaemia (CLL).
Authors: Lane Steven W. , Mollee Peter N. , Bird Robert , Porceddu Sandro , Gill Devinder
Published: April 2020

Aim

The aggressive nature of cutaneous SCC in CLL, specifically presentation with locally invasive, regional metastatic or loco-regionally recurrent disease, is not well documented. We describe a series of patients with CLL who have developed aggressive SCC.

Methods

Patients with CLL with aggressive SCC were identified from existing databases in the Department of Haematology and Oncology, Princess Alexandra Hospital, Australia from August 2001–May 2005. Patient demographics, tumour characteristics and treatment received were reviewed from the clinical records.

Results

We identified 12 patients with CLL with aggressive head and neck SCC: 11 locally invasive, 7 with lymph node metastases and 5 with loco-regional recurrence.

The median age was 73 (range 49–88) and 9 patients were male. 8 patients were Rai stage III or IV. 8 of 9 patients with documented IgG levels had hypogammaglobulinemia (median 3.4g/L). 143 patients with CLL were seen in the hematology department during this time. The median time from CLL diagnosis to SCC development was 55 months (range 0–169). Median time from chemotherapy to aggressive SCC was 48 months (range 22–110) with alkylating agent chemotherapy (seven patients) and 12.5 months (range 1–46) with purine analogue chemotherapy (six patients). Four patients had received no treatment for CLL. All patients had extensive surgical resection and 11 had, or are planned for, post-operative radiotherapy. Five developed loco-regional recurrence at a median of 12 months (range 4–14). 10 patients had numerous SCC (>5) excised prior to the diagnosis of aggressive SCC.

Conclusions

The risk of patients with CLL developing aggressive cutaneous SCC, with its attendent morbidity, is underappreciated. Cellular and humoral immunodeficiencies from CLL and its chemotherapy treatment may predispose patients to aggressive SCC.