bluuebunny/crossref_metadata_2025 huggingface dataset:
Numerous Genomic Abnormalities in AML with Normal Karyotype.
Authors: Akagi Tadayuki , Ogawa Seishi , Yamamoto Go , Nannya Yasuhito , Sanada Masashi , Kawamata Norihiko , Miller Carl W. , Dugas Martin , Schnittger Susanne , Haferlach Torsten , Haferlach Claudia , Koeffler H. Phillip
Published: April 2020

Acute myeloid leukemia (AML) is a clonal hematopoietic malignant disease; about 45–50% of AML samples have no detectable chromosomal abnormality. We wanted to determine if alterations of the genome were present in these AML samples using a sensitive technique and to determine what were these common abnormalities. Thirty-seven normal karyotype AMLs were analyzed with high-density single-nucleotide polymorphism microarray (250K SNP-chip) using the new algorithm AsCNAR (allele-specific copy-number analysis using anonymous references). Nineteen samples (51%) showed either one or more genomic abnormalities including duplication, deletion and loss of heterozygosity with normal copy number [we call this somatic uniparental disomy (UPD)]. Importantly, 12 patients (32%) had UPD, causing duplication of either mutant FLT3 (2 cases), mutant JAK2 (1 case) or mutant AML1/RUNX1 (1 case); and each of these samples had loss of the normal allele. Ten patients (27%) had small copy number changes including deletions of either NF1, ETV6/TEL, or the CDKN2A (p16/INK4A and p14/ARF) and CDKN2B (p15/INK4B) gene complex; these genes can normally function as tumor suppressor genes. mRNA microarray analysis was done on all samples. One case clearly showed a relationship between chromosomal changes and mRNA expression levels: loss or gain of chromosomes leading to decrease or increase mRNA expression of genes; however, in general a loss or gain of a gene could not be detected by microarray analysis. This study suggests that at least one half of AML patients with normal karyotype have readily identifiable genomic abnormalities as found by SNP-chip and AsCNAR. Especially notable is the high frequency of UPD. This technique may become a routine, rapid, robust technique for prognostic stratification of patients and to screen for novel therapeutic targets.
PKCδ Regulates Retinoic Acid-Induced Myeloid Cell Differentiation through Eukaryotic Initiation Factor Alpha (eIF2α).
Authors: Ozpolat Bulent , Akar Ugur , Barria Magaly , Lopez-Berestein Gabriel
Published: April 2020

Overactivity of eukaryotic initiation factor-alpha (eIF2α) has been shown to be oncogenic and induces malignant transformation. Here we investigated the regulation and the role of eIF2α in the terminal differentiation of myeloid leukemia cells. We found that all-trans-retinoic acid (ATRA) and other granulocytic differentiation inducers, such as dimethylsulfoxide and arsenic trioxide inhibited activity of eIF2α by inducing serine 51 phosphorylation in promyelocytic leukemia cells (NB4). In contrast, activity of eIF2α was unaffected during ATRA-induced monocytic differentiation of U937 and THP-1 myelomonocytic cells and phorbol 12-myristate 13-acetate-induced monocytic/macrophagic differentiation of NB4 cells. Knockdown of eIF2α by RNA interference (siRNA) significantly inhibited (p<0.05) ATRA-induced differentiation, indicating that eIF2α is critical for the induction of granulocytic differentiation. ATRA-induced eIF2α phosphorylation was correlated with the expression and activity/phosphorylation (Thr505 and Ser643) of protein kinase Cδ (PKCδ) and eIF2a kinase PKR (Thr446). The specific PKCδ inhibitor Rottlerin significantly reduced phosphorylation of eIF2α, activity of PKR and blocked ATRA-induced granulocytic differentiation of NB4 cells (p<0.05). Knockdown of PKCδ by siRNA decreased PKR activity and increased eIF2α activity while knockdown of PKR increased eIF2α activity. We also observed that PKCδ regulates eIF2a activity in normal CD34+ bone marrow progenitor cells and breast and pancreatic cancer cell lines. Furthermore, we found that eIF2α regulated the expression and activity of important targets of ATRA, including c-myc, p21Waf1/Cip1, DAP5, GADD153, ATF-2, TG2, and p-P70S6K. In conclusion, our findings indicate that ATRA-induced granulocytic differentiation of myeloid leukemia cells is regulated by PKCδ through the activity of eIF2α, revealing a novel mechanism of granulocytic cell differentiation.

Figure Figure
CD3+/CD56+ Cells, but Not Natural Killer T Cells, Are Increased in Peripheral Blood of Untreated Patients with Leukemia.
Authors: Le Dieu Rifca , Taussig David , MacDougal Finlay , Lister Andrew , Gribben John G.
Published: April 2020

Despite recent interest in the use of natural killer T (NKT) cells for cancer immunotherapy, there remains confusion over the phenotype and function of these cells. We observed a population of CD3+/CD56+ cells in the peripheral blood (PB) of patients with acute myeloid leukemia (AML) at diagnosis. We therefore performed more detailed multiparametric flow cytometric analysis on PB from previously untreated patients with AML (n=30), chronic lymphocytic leukemia (CLL) (n=12) and healthy normal volunteers (n=17) to characterize these cells in more detail. Since we were assessing rare cell populations, at least 200,000 events were always acquired from the mononuclear and live cell gates. In healthy individuals CD3+/CD56+ cells comprise 1% to 8.6% (median 2.74%) of PB T cells, but these cells are significantly increased both in percentage of T cells as well as in absolute numbers in the PB of patients with AML (p=0.0009) and CLL (p<0.0001). The percentage of these cells expressing the activation markers CD25 and CD69, is significantly higher in patients with AML (p=0.0007), but not CLL (p=0.67). We next assessed whether there was an increase in true NKT cells identified using anti-CD3 mAb and CD1d molecule loaded with alpha-galactosylceramide tetramer (kindly provided by the NIH tetramer facility). A tetramer consisting of an unloaded CD1d molecule was used as a negative control. Results were confirmed using fluorochrome-labelled antibodies to the T cell receptor chains Valpha24 and Vbeta11. Consistent with previous reports, true NKT cells ranged from undetectable to 1.59% (median 0.03%) of PB T cells in normal individuals. These NKT cells express CD4 or CD8alpha but few express the NK markers CD16 or CD56. There was no significant increase in true NKT cells in percentage or absolute numbers in AML (p=0.56) or CLL (p=0.4), however there are significant differences in subsets of these NKT cells in patients with AML compared with normal individuals. Taken together, these results demonstrate an increase in CD3+/CD56+ cells, but not true NKT cells, in PB of patients with acute and chronic leukemias. CD3+CD56+ cells do not represent NKT cells in health or in disease and true NKT cells require specific characterization. Functional analysis of both CD3+/CD56+ cells as well as true NKT cells in their ability to recognize and lyze AML and CLL cells is underway.
A Chimeric Gene, FIP1L1-RARα, Is Isolated from t(4;17)-Positive Acute Promyelocytic Leukemia; a Report of Cloning and Functional Analysis.
Authors: Kondo Takeshi , Mori Akio , Onozawa Masahiro , Kahata Kaoru , Hashino Satoshi , Tanaka Junji , Imamura Masahiro , Morioka Masanobu , Asaka Masahiro
Published: April 2020

Acute promyelocytic leukemia (APL) comprises 5 to 10% of acute myelogenous leukemia, the leukemia cells of which is characterized by the differentiation block at the stage of promyelocyte. In APL cells, a reciprocal translocation in chromosome 17 is usually observed, and consequently, a retinoic acid receptor alpha (RARα) gene is rearranged. The rearranged RARα gene is fused to another gene, and as a result, the product of newly composed fusion gene provokes the onset of APL. So far, there are five RARα partner genes reported; PML gene in 15q22, PLZF gene in 11q23, NPM gene in 5q35, NuMA gene in 11q13 Stat5b in 17q21. We recently experienced APL case, in which a chromosomal translocation, t(4;17) was observed. FISH analysis of leukemia cells indicated that RARα gene was translocated. To identify the RARα-partner gene, we performed 5′-rapid amplification of cDNA end (RACE) and identified FIP1L1 gene as RARα-partner gene. It is known that FIP1L1 gene is fused to PGDFRα gene in the hypereosinophilic syndrome. DNA sequence revealed that exon 15 of FIP1L1 gene was fused to exon 3 of RARα gene. In previous reports, five X-RARα have the common character;

X-RARα manifests homodimerization. X-RARα suppresses retinoic acid-induced transcription.

We examined that FIP1L1-RARα has these two characters. Transient transfection analysis showed that FIP1L1-RARα has the ability to form homodimer. And luciferase assay suggested that FIP1L1-RARα suppressed the retinoic acid-induced transcriptional activity at the physiological concentration of all-trans retinoic acid (ATRA). And the level of luciferase activity suppressed by FIP1L1-RARα was similar to that suppressed by PML-RARα. And the therapeutic concentration of ATRA activated the significant level of luciferase activity. Thus, in vitro analysis suggested that ATRA treatment could be effective to FIP1L1-RARα-positive APL patients. Consistent with the results of in vitro analysis, ATRA treatment was clinically effective, and the patient achieved complete remission after five weeks of ATRA treatment. Collectively, we suggest that FIP1L1-RAR? is the sixth pathogenic gene of APL.
Etoposide Sensitivity Does Not Predict MLL Rearrangements or Risk of Therapy-Related Acute Myeloid Leukemia.
Authors: Yang Jun , Bogni Alessia , Cheng Cheng , Bleibel Wasim K. , Cai Jean , Fan Yiping , Yang Wenjian , Rocha Jose Claudio , Pei Deqing , Liu Wei , Dolan M. Eileen , Pui Ching-Hon , Relling Mary V.
Published: April 2020

Therapy-related acute myeloid leukemia (t-AML) can arise from topoisomerase II-directed agents such as etoposide and teniposide, most likely via drug-induced MLL gene fusions. MLL cleavage in its breakpoint cluster region has been linked to apoptosis caused by etoposide and other agents. It has been speculated that the degree of myelotoxicity caused by agents such as etoposide may predispose to t-AML. However, whether drug-induced MLL rearrangements (rMLL) and subsequent leukemogenesis are inextricably linked to the desired drug-induced cytotoxic effects remains a matter of uncertainty. To directly address this question, we compared rMLL in blood samples from and epipodophyllotoxin-related clinical toxicity in children with acute lymphoblastic leukemia (ALL) who did and did not develop t-AML, as well as the level of rMLL in cell lines that were sensitive or resistant to etoposide. In 7 t-AML cases and 7 identically-treated matched controls, rMLL were detectable following etoposide treatment in both cohorts, but at a slightly greater frequency in the former group (P = 0.04, Wilcoxon signed-rank test). However, there was no correlation between the cumulative etoposide dose and the degree of rearrangements (P = 0.5, r2=0.039, Spearman rank correlation). Secondly, etoposide or teniposide-related acute toxicities to the host tissues did not differ between the 14 t-AML cases and the 14 matched controls. The drug-induced reduction in neutrophil and leukocyte counts was not greater among t-AML cases than controls (P>0.17, Wilcoxon signed-rank test), nor was the incidence of infection and gastrointestinal toxicity during epipodophyllotoxin treatment (P>0.22, McNemar’s test). Finally, in 25 human lymphoid cell lines, MLL fusions were more common after etoposide treatment (at equitoxic concentrations) compared to untreated controls (P < 0.0001, paired t test) but did not differ in frequency in etoposide-sensitive versus resistant cell lines (P = 0.91, Mann-Whitney U test). Together, these data indicate that epipodophyllotoxin-mediated leukemogenesis is not directly linked to the cytotoxicity of these agents.
Elderly Patients with Acute Myeloid Leukemia Are Not All the Same: Results of the Prospective DSIL AML96 Trial.
Authors: Schaich Markus Andreas , Aulitzky Walter E. , Bodenstein Heinrich , Bornhaeuser Martin , Illmer Thomas , Schaekel Ulrike , Schmitz Norbert , Schuler Ulrich S. , Soucek Silke , Thiede Christian , Wandt Hannes , Ehninger Gerhard
Published: April 2020

The majority of patients with acute myeloid leukemia (AML) are older than 60 years at diagnosis. However, treatment results for these elderly patients are still unsatisfactory. This is thought to be due to a more aggressive disease, preexisting co-morbidities or a decreased tolerance for intensive treatment approaches. As for younger patients there is growing evidence that elderly AML patients may be divided into prognostic subgroups. So far data on prognostic factors in this group of patients are still sketchy. Between February 1996 and March 2005 a total of 827 elderly AML patients with a median age of 67 (61–87) years were treated within the prospective AML96 trial of the German Study Initiative Leukemia (DSIL). 643 patients had de novo and 184 patients secondary disease. All patients were scheduled to receive a double induction therapy with Daunorubicin and Ara-C (DA3+7). The consolidation therapy consisted of one course of m-Amsacrine and intermediate-dose (10g/m2) Ara-C. 265 (32%) patients reached CR criteria after double induction therapy. Forty-two patients (5%) had only a PR, 307(37%) displayed refractory disease, 126(15%) died during induction therapy and 77(10%) received only one course of induction therapy due to severe toxicity. Out of the 265 patients in CR 120 (45%) patients received the consolidation course. The strongest independent prognostic factors for achieving a CR were less than 10% blasts in the day 15 bone marrow, the presence of a NPM mutation or a low-risk karyotype (p<0.0001 each). The 3-year overall (OS) and relapse-free survival (RFS) rates were 18% for all patients and 17% for all patients in CR, respectively. In the multivariate analysis the strongest prognostic factors for survival were age, LDH and cytogenetics (p<0.0001 each). Using these three parameters a prognostic model for survival was established. Patients older than 70 years with intermediate- or high-risk cytogenetics and a high LDH level at diagnosis (n=213) had a 3-year OS of only 9%, whereas patients with low-risk cytogenetics or patients with intermediate-risk cytogenetics, younger than 70 years and a low LDH level (n=237) had a 3-year OS of 32%. All other patients (n=377) had an intermediate 3-year OS of 15% (p<0.0001). In conclusion, elderly AML patients can be stratified into prognostic groups. AML patients older than 70 years with high LDH levels and intermediate- or high-risk cytogenetics at diagnosis do not profit from conventional chemotherapy.
Venous Thromboembolic Risk and Suboptimal Prophylaxis in the US Acute Hospital Care Setting: Findings from the ENDORSE Study.
Authors: Tapson Victor F. , Cohen Alexander T. , Bergmann Jean-Francois , Goldhaber Samuel , Kakkar Ajay K. , Anderson Frederick A.
Published: April 2020

Introduction Estimates for patients at risk of venous thromboembolism (VTE) in US acute care hospitals total approximately 4 million surgical and 8 million medical patients annually. However, limited data are available regarding practices in VTE prevention in the acute care setting. The ENDORSE (Epidemiologic International Day for the Evaluation of Patients at Risk of Venous Thrombosis in the Acute Hospital Care Setting) study is a worldwide survey of inpatients in acute hospital care. We assessed data from US acute care hospitals to determine the prevalence of VTE risk and proportion of at-risk patients receiving prophylaxis in accordance with current ACCP guidelines.

Methods During the period August 2006 to January 2007, 9257 patients were enrolled from 81 randomly-selected US acute care hospitals. All medical patients ≥40 years, surgical patients ≥18 years, or patients ≥18 years admitted for non-surgical trauma were eligible for the study. Patients admitted to an ineligible ward, with missing data, or admitted solely for the treatment of VTE were excluded from the analysis. Patient charts were reviewed during a single day, pre-defined visit, with the evaluation criteria including medical history, admission and post-admission diagnoses, type of surgery, bleeding risks, and initiation and type of prophylaxis. The ACCP consensus guidelines were used to evaluate VTE risk and prophylaxis use in enrolled patients.

Results Of the 9257 study patients, 44% and 56% were surgical and medical, respectively. 78% and 52% of surgical and medical patients were judged at risk for VTE, respectively. Of these, 29% of surgical patients and 52% of medical patients failed to receive recommended types of prophylaxis (Table). Among at-risk patients who received recommended types of VTE prophylaxis, a majority received anticoagulants (90% surgical, 97% medical).

Table: VTE risk and prescription of recommended types of prophylaxis in US acute care hospitals US patients [X, range*] At-risk for VTE [X, range*] At-risk patients who received ACCP-recommended prophylaxis [X, range*] *Mean and range across all 81 US hospitals All (N=9257) 5885 [64%] 3536 [60%] Surgical (N=4061) [44%,13–94] 3165 [78%, 21–100] 2244 [71%, 0–100] Medical (N=5196) [56%, 6–87] 2720 [52%, 19–100] 1292 [48%, 0–100]

Conclusions These data from US acute care hospitals parallel the global ENDORSE study findings of the high prevalence of VTE risk and suboptimal provision of ACCP-recommended prophylaxis. Although a higher proportion of surgical patients were judged to be at risk of VTE, the absolute number of hospitalized medical patients was higher and half of these did not receive adequate VTE prophylaxis. Our data highlight the continued need for strategies to ensure that hospital patients at risk of VTE are identified and provided with adequate VTE prophylaxis.
Argatroban Associated Bleeding Complications.
Authors: Berz David , Colvin Gerald A. , Mital Priya , Quesenbery Peter J.
Published: April 2020

Background: Heparin induced thrombocytopenia (HIT) is a frequently observed side effect observed with heparin administration. Treatment for this condition includes direct thrombin inhibitor (DTI) therapy. Major hemorrhagic complications are considered rare events in this setting.

Methods: This is a case series of patients diagnosed with HIT in a tertiary medical referral center. The diagnosis of HIT was established on the base of clinical criteria. All included patients had positive HIT ELISA based antibody testing during the time interval from July 2006 to July 2007 and all patients were treated with argatroban. A systematic review for hemorrhagic and thrombotic complications of argatroban therapy was performed. Major hemorrhage was defined as clinically identified bleeding with the demand for transfusion therapy. Thrombotic complications were defined as thrombosis evidenced by imaging studies developing during argatorban therapy. We performed bivariate and multivariate logistic regression analysis to identify demographic and clinical factors that influence the development of complications of argatroban therapy as treatment for HIT. Examined covariates were gender, age, type of service the patient was admitted under (surgical versus medical), amount of comorbidities, preexisting history of gastrointestinal bleeding, documented thrombotic or thrombembolic event at the time point of HIT diagnosis and coexisting coexisting coagulopathy other than HIT.

Results: We identified 102 patients with the diagnosis of HIT. The median optical density (OD) of the ELISA HIT immunoassay was 1.23 (95% CI 0.93–1.43). We identified 11 (10.7%) major hemorrhagic events in patients on argatroban. Four patients (3.9%) died as a consequence of major hemorrhagic complications. No thrombotic events were identified in our study cohort. As statistically significant; predictors for clinically relevant hemorrhagic complications in our model remained male gender, preexisting GI bleeding history and being patient on a surgical service.

Conclusion: The incidence of major hemorrhagic episodes with agatroban therapy is significantly higher then generally reported and the mortality rate is very disturbing. These data suggest that alternatives to standard direct thrombin inhibitor therapy should be aggressively pursued.
Periostin Is a Previously Uncharacterised Vitamin K Dependent γ-Carboxyglutamic Acid (Gla) Containing Protein Expressed by Marrow-Derived Mesenchymal Stromal Cells.
Authors: Coutu Daniel L. , Wu Jian Wui , Rivard Georges-Etienne , Blostein Mark D. , Galipeau Jacques
Published: April 2020

The modification of glutamic acid residues to g-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent γ-glutamylcarboxylase enzyme. Despite ubiquitous expression of the γ-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-proteins after the liver, we sought to identify Gla proteins secreted by bone-marrow derived mesenchymal stromal cells (MSCs), a precursor to all non-hematopoietic cells in bones. We used a proteomics approach to screen the secretome of MSCs with a combination of 2D gel electrophoresis and tandem mass spectrometry. The most abundant Gla-protein secreted by MSCs was identified as periostin, a previously unrecognized γ-carboxylated protein. In silico aminoacid sequence analysis of periostin demonstrated the presence of four consensus γ-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation using anti-Gla antibodies and could be inhibited by warfarin in MSCs. In conclusion, periostin is a novel vitamin K-dependent γ-carboxylated protein distinguished from other Gla-proteins by the presence of multiple γ-carboxylase recognition sites and MSCs are an abundant source of periostin including its γ-carboxylated variant.
An Intron-Derived Insertion/Truncation Mutation in the BCR-ABL Kinase Domain in Three CML Patients Undergoing Kinase Inhibitor Therapy.
Authors: Laudadio Jennifer , Deininger Michael W.N. , Mauro Michael J. , Druker Brian J. , Press Richard D.
Published: April 2020

Although targeted inhibition of BCR-ABL with imatinib is an effective therapy for patients with chronic myeloid leukemia, a minority acquire mutations in the kinase domain (KD) that cause imatinib resistance. The spectrum of KD mutations thus far discovered, although quite heterogeneous, includes almost exclusively single nucleotide substitutions in key amino acids regulating drug binding or BCR-ABL function. Here, we describe a KD insertion/truncation mutation in 3 CML patients undergoing kinase inhibitor therapy. Two of these patients were being treated with imatinib (for 12 and 17 months), and one with dasatinib (for 13 months after a prior relapse while on imatinib). Suspected drug resistance was assessed by direct DNA sequencing of a BCR-ABL PCR product extending to the end of the kinase domain. Each of these 3 patients had 35 nucleotides from ABL intron 8 inserted at the normal exon 8–9 splice junction, after nucleotide 1423 (amino acid 475) of Genbank cDNA clone NM_005157. In all 3 cases, the mutation was co-expressed with wild type BCR-ABL sequence. The inserted sequence is derived from intron 8, beginning 1151 bp downstream from the normal splice donor site at the end of exon 8. This 35 bp intronic sequence is flanked by excellent consensus splice donor and acceptor sequences, suggesting alternative splicing as the likely mutational mechanism. The insertion creates a premature translational stop codon after 10 intron-encoded amino acids (figure), thus truncating 653 C-terminal amino acids including part of the KD and the entire last exon region - including a proline-rich domain, 3 nuclear localization signals, a DNA-binding domain, an actin-binding domain, and a nuclear export signal. These 3 insertion mutation cases were detected in our diagnostic clinical molecular pathology laboratory after sequencing 174 cases referred to us for suspected kinase inhibitor resistance, 78 of which contained a detectable mutation. The estimated prevalence of the exon 8/9 insertion/truncation mutation is then approximately 1.7% among patients with suspected drug resistance, and this mutation constitutes approximately 3.8% of all mutations.

Conclusion: Kinase domain insertions are an alternative (and not entirely uncommon) mutational mechanism in CML patients undergoing kinase inhibitor therapy. The functional significance in terms of kinase activity and drug resistance remains to be addressed.

Figure: Amino acid sequence of the C-terminus of the BCR-ABL kinase domain for the wild type and insertion/truncation mutant (with numbering as per GenBank cDNA clone NM_005157). Figure: Amino acid sequence of the C-terminus of the BCR-ABL kinase domain for the wild type and insertion/truncation mutant (with numbering as per GenBank cDNA clone NM_005157).
Dasatinib Resistance in CML Patients. Identification of Novel BCR-ABL Kinase Domain Mutation.
Authors: Krijanovski Yelena , Donato Nicholas , Quintas-Cardama Alfonso , Cortes Jorge , Talpaz Moshe
Published: April 2020

Dasatinib is a dual specific SRC-ABL kinase inhibitor which has been increasingly used in treatment of CML and Ph + ALL patients. Although it has excellent activity, a proportion of patients, particularly those with advanced disease had developed evidence of dasatinib resistance. In order to assess the reasons for resistance we have analyzed BCR-ABL kinase domain utilizing single step PCR reaction followed by amplification of ABL kinase domain with subsequent subcloning (≥10 clones) from 23 CML patients who demonstrated clinical evidence of dasatinib failure. We have found mutations in the BCR-ABL domain in 19 out of 23 patients. 14 were mapped to ATP “binding pocket”, of those, seven had pan-resistant T315I and five had F317L with one F317I mutation. In addition, three of 19 patients had V299L mutation. All of these mutations were previously reported in association with dasatinib resistance. We also observed that one of 8 patients in chronic phase and three of 15 patients in advanced phase had two different mutations in separate clones§. Interestingly, four of 19 patients were found to have novel V304D mutation, which is located outside of dasatinib binding site in β 4–5 loop structure and may not be directly involved in dasatinib resistance. It appears that BCR-ABL mutations in advanced phase CML are more common, but occur at lower frequency in dasatinib resistant patients in chronic phase, 93% vs. 62%. In conclusion, our clinical sample suggests that selection for T315I and F317L mutations occurs frequently in dasatinib resistant patients which was predicted from preclinical study. Both mutations were proposed to directly and indirectly affect dasatinib binding. Two additional mutations have been identified with incidence that exceeds random occurrence. The significance of these mutations (V299L, D304D) will be further studied.

Table 1. Mutations status at different phases of CML chronic phase advanced phase mutations yes 5 § no 3 yes 14§ no 1 1 patient in chronic phase group have subcloning results pending. T315I 2 7 F317L 1 4 F317I 0 1 V299L 1 2 V304D 1 3
Incidence and Clinical Outcome of Intestinal Thrombotic MicroAngiopathy (TMA) in Patients Suspected to Clinical Graft Versus Host Disease (GVHD).
Authors: Park Gyeongsin , Moon Yeonsook , Eom Ki-Seong , Lee Kyungji , Lee Youn-Soo , Kim Yoo-Jin , Kim Yonggoo , Cho Bin , Cho Seok-Goo , Kang Chang-Suk , Lee Kyo-Young
Published: April 2020

Intestinal TMA, a upcoming and serious problem after HSCT, usually manifests diarrhea, vomiting, GI bleeding and abdominal pain as initial clinical presentations similar to those of intestinal GVHD. But intestinal TMA should be distinguished from GVHD, because they needed to be treated in absolutely different way. Here we investigated the incidence of intestinal TMA and evaluated their clinical progressions in HSCT patients. We retrospectively reviewed 243 gastrointestinal mucosal biopsy slides obtained from HSCT patients having suffered from GI problems in the Catholic University Hospital, from 2000 to 2004. The collected cases were reviewed by two pathologists blindly and classified into TMA; showing microthrombi formation in the lumina of microvasculatures, GVHD; showing epithelial apoptosis and/or loss of glands, TMA+GVHD and non-specific inflammation. And next, their incidence and clinical outcomes were analyzed. On histopathological examination, 15 out of 243 cases (6.2%) showed luminal thrombi of microvasculature compatible with TMA, 58 cases (23.9%) showed findings compatible with GVHD, 3 cases (1.2%) showed both TMA and GVHD, and 173 cases (71.2%) showed nonspecific gastroenteritis. On peripheral blood smear examination, all 15 TMA cases showed less than 1% of fragmented RBC at the time of biopsy. But 2 out of 15 cases (13.3%) showed increase to 8% and 10% of fragmented RBC in follow-up, and they had been aggravated clinically and improved only after treatments including discontinuing immunosuppressant and plasma exchange. Among 58 GVHDs, 34 patients (75.9%) were improved with steroid pulse therapy, but 14 patients (24.1%) failed. These results show that intestinal TMA is not infrequent in HSCT patients and can be combined with intestinal GVHD. In conclusion, intestinal TMA should be recognized in pathological diagnosis of HSCT patients. It is important to differentiate intestinal TMA from GVHD in patients suffering from severe and refractory diarrhea after HSCT.
Elevated CD56dimCD16neg NK Cells on Day 28 after HCT: Role in Immunity Against Viral Infections or Residual Tumor Cells?.
Authors: Khan Faisal , Storek Jan
Published: April 2020

Natural killer (NK) cells represent a lymphocyte subset important in the immune surveillance against virus infected and malignant cells. In humans, two major NK cell subsets have been defined by CD56 and CD16 staining:

Regulatory NK cells (CD56 high CD16 neg), which are potent cytokine (IFNg) producers; and Cytolytic NK cells (CD56 dim CD16 pos) that are involved in target cell lysis.

Functions of other NK cell subset are unknown. One of such subset, CD56 dimCD16 neg NK cells have recently been reported to represent cytolytic cells, degranulated after contact with target cells. NK cells are the first lymphocyte subset recovering to normal quantity and function after an allogeneic or autologous HCT, typically by 4 weeks post HCT. In the present investigation, we attempted to determine percentages of following NK cell subsets (Regulatory NK cells, Cytolytic NK cells, CD56dimCD16 neg, CD56 highCD16 pos and CD56neg CD16 high NK cells) at multiple time points (day 28, 56, 84, 180 and 365) after allogeneic (n= 32) and autologous (n= 25) HCT and compared with that of healthy individuals (n=14). Whole blood specimen was incubated with fluorochrome-conjugated monoclonal antibodies (CD3-ECD, CD14-PE, CD56-FITC and CD16-PC5) and analyzed by four color flowcytometry. NK cells were defined as mononuclear cells expressing CD16 or CD56 and not expressing CD3 or CD14. The most striking finding was that of significant increase in percentage of CD56dimCD16−ve cells (among NK cells) on day 28. This was true for both autologous HCT recipient (n=24; median = 1.02% of total NK cells) and allogeneic HCT recipient (n=26; median = 1.1% of total NK cells) in comparison to the healthy individuals (n=14; median = 0.56% of total NK cells). We speculate the rise in CD56dimCD16−ve NK cells at 1 month post HCT may be due to increased degranulation of cytolytic NK cells in response to viral infections or residual malignancy.
BMP and Wnt Induce Embryonic Blood Fate by Activation of the Cdx-Hox Pathway.
Authors: Lengerke Claudia , Schmitt Sabine , Bowman Teresa , Maouche-Chretien Leila , McKinney-Freeman Shannon L. , Jang Il Ho , Davidson Alan J. , Hammerschmidt Matthias , Rentzsch Fabian , Green Jeremy B. , Zon Leonard I. , Daley George Q.
Published: April 2020

The hematopoietic morphogen BMP4 has been implicated in early blood development, however the precise role and the underlying genetic pathways that link BMP action to blood fate specification remain poorly understood. In this study, we have employed the in vitro system of murine embryonic stem (mES) cell differentiating into embryoid bodies (EBs) as a model to explore mesoderm induction and hematopoietic specification during mammalian embryogenesis. We show that BMP plays a role in blood fate specification in two sequential phases: initially, BMP acts as a dorsoventral patterning agent, inducing ventroposterior mesoderm; at a later phase, BMP is required for hematopoietic fate specification from mesoderm. In dissecting this second specification phase, we discovered a previously unsuspected coordination between BMP4 and Wnt3a signaling, and linked these morphogens directly to Cdx and Hox genes. Using chromatin immunoprecipitation assays performed on EB-derived cells, we detected the association of the Wnt signaling effector LEF1 with the proximal Cdx1 promoter. This association was modulated by BMP signaling. In promoter reporter assays both BMP4 and Wnt3a activated the Cdx1 promoter while Wnt inhibitors or point mutations in the LEF1 responsive elements of the Cdx1 promoter abrogated this response. Together, these results suggest that BMP4 activates the Cdx1 promoter via LEF1 and the Wnt pathway. In functional assays, inhibition of Wnt signaling strongly suppressed the formation of hematopoietic colony initiating cells, suggesting that activation of the Wnt-Cdx-Hox pathway is a central downstream component of the BMP dependent blood fate specification program. Using ES cell lines engineered for conditonal Cdx1 or Cdx4 gene overexpression, we demonstrate that ectopic activation of Cdx genes is sufficient to establish hematopoietic fate decisions in the absence of BMP signaling during mesoderm specification to blood. Finally, to explore the relevance of our findings in vivo, we studied the effects of conditional BMP overexpression on cdx4 expression and embryonic blood formation during zebrafish embryogenesis. Matching our results with EBs, we find that overexpression of bmp2b at midgastrulation expands the cdx4 expression domain and strongly enhances blood formation. Taken together our results reveal an interaction between the BMP and Wnt signaling pathways, upstream of the Cdx genes, that is critical for the induction of the embryonic blood cell program. Given the evolutionary conservation of the BMP-Wnt-Cdx-Hox effect on blood formation from fish to mice, we anticipate that similar pathways can be exploited to direct the differentiation of human ES cells into blood.
Cord Blood Transplantation from Unrelated Donors for Children with Acute Lymphoblastic Leukemia in Japan: The Impact of Methotrexate on the Clinical Outcome.
Authors: Kato Koji , Yoshimi Ayami , Ito Etsuro , Oki Kentaro , Hara Jun , Nagatoshi Yoshihisa , Kikuchi Akira , Kobayashi Ryoji , Nagamura-Inoue Tokiko , Kai Shunro , Azuma Hiroshi , Takanashi Minoko , Isoyama Keiichi , Kato Shunichi
Published: April 2020

Cord blood transplantation (CBT) became one of the important alternatives in allogeneic stem cell transplantation for children with hematological malignancies. We have analyzed the clinical outcomes of CBT for children with acute lymphoblastic leukemia (ALL) in Japan and identified risk factors of transplant outcome, when they had no prior transplant. From 1997 to 2006, total 332 children with ALL have undergone CBT from unrelated donor and 270 of them had no prior transplant. They are 0–15 yrs (median 5) and 4 to 60kg of body weight (median 18). Serological disparities of HLA for graft-versus-host disease (GVHD) direction were 0(n=54), 1(n=168) and 2(n=47), and disease status at transplant were 1st complete remission (CR) (n=120), 2nd CR (n=71), and more advanced stages (n=75). The median number of nucleated cells in cord blood unit was 4.93×107/kg (1.35–24.9), and that of CD34+ cells was 1.53×105/kg (0.17–15.0). As preconditioning, total body irradiation (TBI) was given in 194 patients and methotrexate (MTX) was given as GVHD prophylaxis in 159 patients. The neutrophil engraftment was achieved in 88.5% (95%CI: 84.1–91.8%) of patients and platelet engraftment (>50k) was obtained in 72.6% (95%CI: 66.8–77.7). The incidence of grade II–IV and III–IV acute GVHD was 45.6% (95%CI: 39.5–51.4) and 20.4% (95%CI: 15.8–25.4) respectively. Non-relapse mortality was observed in 22.6% (95%CI: 17.7–27.8) and 35.2% (95%CI: 29.2–41.3) of patients relapsed after CBT. The five year event free survival (EFS) of all patients was 38.1% (95%CI: 34.9–41.3); 47.4%(95%CI: 42.4–52.4) in 1st CR, 45.5%(95%CI: 38.9–52.1) in 2nd CR and 15.2%(95%CI: 10.8–38.9) in more advanced stages, respectively. The multivariate analysis revealed that the larger number of CD34+ cells (p<0.01) and administration of granulocyte colony stimulating factor after CBT (p<0.01) were associated with earlier neutrophil engraftment. Preconditioning with TBI (p<0.01) and absence of MTX (p=0.037) significantly affected the development of grade II-IV acute GVHD. Advanced disease at transplant was the most predominant factor for leukemic relapse (p<0.01). GVHD prophylaxis with MTX (p=0.042), less allele mismatches (p=0.013) and disease status of 1st and 2nd CR at transplant (p<0.01) were significantly associated with better EFS. Our results showed the favorable effect of MTX for the development of acute GVHD and EFS. In conclusion, GVHD prophylaxis including MTX is important in CBT for children with ALL.
Unrelated Donor Umbilical Cord Blood Transplantation (UD-UCBT) for Adult Patients with High-Risk Acute Lymphoblastic Leukemia (ALL).
Authors: Sanz Jaime , Lorenzo Ignacio , Montesinos Pau , Senent Leonor , Larrea Luis , Planelles Dolores , Martin Guillermo , Cervera Jose , Jarque Isidro , de la Rubia Javier , Orti Guillermo , Scaff Miguel , Paciello Mari Liz , Moscardo Federico , Romero Monica , Biologist Luis Benlloch , Sanz Miguel A. , Sanz Guillermo
Published: April 2020

BACKGROUND: Adult patients with high-risk ALL can benefit from allogeneic stem cell transplantation but most lack a suitable sibling or unrelated adult donor. Umbilical cord blood has emerged as an alternative source for stem cell transplantation.

OBJECTIVES: The aim of the present study was to evaluate toxicity and efficacy of UD-UCBT for the treatment of high-risk ALL in adults as well as to identify by multivariate analysis factors affecting transplant outcome.

PATIENTS AND METHODS: Thirty-seven consecutive patients (23 males, 14 females) with median age 26 yr (range, 15–47) who underwent UD-UCBT at a single institution from 1999 until 2006 were analyzed. Primary high-risk feature was Philadelphia-positive ALL (12), MLL rearrangement (3), primary refractory disease (3), salvage treatment for patients in second complete remission (CR2, 4) or beyond CR2 (6), and slow response to initial therapy or 2 cycles to achieve CR (9). Conditioning regimen consisted of thiotepa, busulfan (oral, 13; IV, 24), cyclophosphamide (24) or fludarabine (13), and anti-thimocyte globulin. Cyclosporine and prednisone were used for graft-versus-host disease (GVHD) prophylaxis. Most patients (95%) received an HLA-mismatched cord blood unit with 1 (35%) or 2 (60%) HLA disparities. At infusion, the median number of nucleated cells and CD34+ cells was 2.4 × 107/kg and 1.3 × 105/kg, respectively.

RESULTS: Cumulative incidence (CI) of myeloid and platelet engraftment was 92% and 76% at a median time of 21 and 63 days respectively. CI of GVHD acute grades II-IV, III-IV and chronic extensive was 29%, 19% and 19% respectively. Transplant-related mortality at 180 days was 29% and was significantly increased in patients developing severe acute GVHD (96% vs 19%; p < 0.001). With a median follow-up of 23 months (range, 7–88), CI of relapse was 34% at 3 years and was higher in patients beyond CR1 (63% vs 28%; p < 0.001). 3-year event-free survival (EFS) and overall survival (OS) were 33% and 37%, respectively. Patients in CR1/2 had better EFS (41% vs 14%; p = 0.04) and OS (50% vs 14%; p = 0.04) while those who developed severe acute GVHD had a worse EFS (42% vs 0%; p = 0.004) and OS (49% vs 0%; p = 0.004).

CONCLUSIONS: These results show that UD-CBT is a curative alternative for a significant number of patients with high-risk ALL. This option should be offered to patients early in the course of their disease to improve outcomes.
Encouraging Results of Reduced Intensity Conditioning (RIC) Umbilical Cord Blood (UCB) Transplantation for the Treatment of Advanced Lymphoid Malignancies.
Authors: Brunstein Claudio G. , Cantero Susana , Cao Qing , Majhail Navneet , McClune Brian , Burns Linda J. , Tomblyn Marcie , Miller Jeffrey S. , Blazar Bruce R. , McGlave Philip B. , Weisdorf Daniel J. , Wagner John E.
Published: April 2020

We have reported on the safety and efficacy of RIC UCB transplantation for patients with advanced hematologic diseases. Here we report on 65 patients, median age of 46 yrs (range, 6–68), who underwent RIC UCB transplantation for the treatment of advanced lymphoid malignancies between October 2001 and December 2006. Patients received either one (n=9) or two (n=56) UCB unit grafts matched at 4–6/6 HLA loci. Forty were male and 27 CMV seropositive. Diagnoses were follicular lymphoma (FL, n=11), small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL, n=9), mantle cell lymphoma (MCL, n=8), large cell lymphoma (LCL, n=14), and Hodgkin’s lymphoma (HL, n=23). Conditioning regimen was cyclophosphamide 50 mg/kg (Day-6), fludarabine 40mg/m2 for 5 days (Day-6 to -2), and single fraction total body irradiation of 200cGy (Day -1), plus cyclosporine A and mycophenolate mofetil (to day +30). At transplantation 53 patients (81%) had chemotherapy sensitive disease, 5 (8%) had bulky adenopathy (≥ 5 cm), 27 (42%) had prior radiation therapy, 26 (40%) had prior autologous transplant, 22 (34%) had an elevated LDH, and 10 (15%) had marrow involvement. The median number of prior treatment courses was 4 (range, 1–9). The median follow-up for survivors is 23 mos. (range, 4–62). Patients were analyzed as indolent (IND=FL+ SLL/CLL), aggressive (AGR=LCL+MCL), and HL. Incidence of acute GVHD grades II–IV was 57% (95%CI, 43–70); grades III–IV 25% (95%CI, 14–35); chronic GVHD 23% (95%CI, 11–35), with similar incidence of acute and chronic GVHD between lymphoma subgroups. The 3-year nonrelapse mortality (NRM) was 15% (95%CI, 5–26) with no significant difference among lymphoma subgroups (IND 5% vs. AGR 24% vs. HL 13%, p=.41). Thirty-five patients (53%) had relapsed or progressive disease with a 3-year progression-free survival (PFS) of 31% (95%CI, 19–44); IND 44% (95%CI, 22–66); AGR 20% (95%CI, 2–39); HL 35% (95%CI, 15–54) (p=.30). Fourteen of these 35 patients were treated with tapering of immunosuppression and rituximab and/or chemotherapy or radiation therapy, and 8 achieved a complete remission. Three-year OS was 55% (95%CI, 42–68); IND 69% (95%CI, 48–90); AGR 54% (95%CI, 33–75); HL 43% (95%CI, 18–69) (p=.37). We conclude that RIC UCB is effective alternative for the treatment of patients with advanced lymphoid malignancy resulting in acceptable NRM and encouraging OS and PFS. Patients with advanced lymphoid malignancies should be considered for a RIC UCBT as a promising alternative for those with no available sibling donor.
Genetic Interaction between PGE2 and the Wnt/β-Catenin Signaling Pathway Regulates Definitive HSC Development and Homeostasis.
Authors: North Trista E. , Goessling Wolfram , Lord Allegra M. , Stoick Cristi , Moon Randall T. , Zon Leonard I.
Published: April 2020

Hematopoietic stem cells (HSC) differentiate throughout the lifetime of the organism to produce each of the definitive blood lineages. Recently, we demonstrated that prostaglandin (PG) E2 regulates vertebrate HSC formation and homeostasis. The mechanism by which PGE2 influences HSCs is currently unknown. Through qPCR analysis and in situ hybridization, we determined that dimethyl (dm) PGE2 regulated expression of the classical wnt targets cyclinD1 and ephrinB2 during embryonic development. As the wnt pathway has been shown to regulate HSC self-renewal, we hypothesized that the two pathways interact genetically to control the production and homeostasis of HSCs. In TOP:dGFP wnt reporter fish, wnt activity was enhanced in vivo following the addition of dmPGE2, whereas indomethacin treatment resulted in the virtual absence of β-catenin/TCF signaling. Activation of wnt signaling in heat-shock (hs) inducible transgenic zebrafish (hs:wnt8) led to enhanced AGM HSC formation, while inhibition of wnt/β-catenin signaling at the membrane level (hs:dickkopf (dkk)), in the cytosol (hs:axin) or in the nucleus (hs:dominant negative (dn) TCF) reduced HSC numbers. Exposure to dmPGE2 rescued HSC inhibition mediated by dkk, but not by axin or dnTCF, implying that PGE2 interacts with the wnt signaling pathway at the level of the β-catenin destruction complex. Furthermore, indomethacin treatment diminished the effects of wnt induction on HSC formation, suggesting that PGE2 activity is required to mediate the full effects of wnt activation. We next sought to determine if PG signaling could regulate wnt-mediated control of hematopoietic recovery in the adult zebrafish following irradiation. As seen with dmPGE2 treatment, wnt induction during marrow recovery led to a significant increase in hematopoietic progenitors by day 10 post injury. Exposure to indomethacin blocked the wnt-mediated increase in kidney marrow precursors. Wnt activation has further been implicated in leukemogenesis. Repetitive activation of wnt signaling in adult fish induced a myeloproliferative disease. Significantly, treatment with indomethacin altered its development and progression. Finally, we investigated the effects of PGE2 modification of wnt activity on hematopoietic homeostasis in the mouse. Following myeloablative 5-FU treatment, PGE2 was capable of modifying wnt-mediated effects on hematopoietic recovery. Compared to wild-type control siblings APCMin mice exhibited elevated numbers of stem and progenitor cells after 2 weeks; treatment with indomethacin every other day prevented this regenerative advantage. These results suggest that pharmacological manipulation of wnt activity through modulation of PGE2 signaling will provide novel therapeutic approaches to regulate HSC homeostasis during bone marrow recovery and leukemogenesis.