bluuebunny/crossref_metadata_2025 huggingface dataset:
Frequency and Dynamics of Bcr1/Bcr3 Isoforms of PML/RARA Fusion Gene - Results of Russian APL Multicenter Trial.
Authors: Savchenko Valeri , Parovitchnikova Elena , Demidova Irina , Shuravina Elena , Isaev Valentin , Miyurin Andrey , Kulikov Sergei
Published: April 2020

Detection and monitoring of minimal residual disease (MRD) in APL patients has become a routine practice and is essentially important for tailoring the therapeutical intervention. Two major isoforms of PML-RARa fusion gene (bcr1/2 and bcr3) are considered to determine diverse outcome with bcr3 to be less favorable. It was also shown by different study groups that among different ethnic populations the ratio of bcr1/2 and bcr3 varies. The prevalence of bcr1/2 isoform over bcr3 was reported in Latinos and Chinese population: 84% vs 16% and 73% vs 27%, respectively. Less evident but nevertheless convincing tendency was registered in USA (US Intergroup – 63% vs 37%; Memorial Sloan Kettering CRC – 66% vs 34%), Great Britain (MRC – 61% vs 39%), Italy (GIMEMA – 60% vs 40%), Spain (PETHEMA – 56% vs 44%) (Santillana S. et al Joint International Congress on APL, Rome, 2001, abstr P2.14; Dan Douer et al BJH, 2003, p.563–570). The analysis of PML-RARa transcript in 66 APL patients included in the Russian Multicenter trial revealed the inverse (statistically significant from mentioned above) ratio with 37 patients (56%) bearing bcr3 isoform and 29 (44%) – bcr1/2. There is no exact explanation for these findings but it may be due to low numbers of cases and perhaps to certain environmental and/or ethnic reasons. The treatment results in these 66 APL patients are very similar to usually reported with 7+3+ATRA protocols – 91% CR rate with 75% molecular CR after the first and 96% - after the second course. The OS and DFS do not differ according to the isoform and constitute 77% and 80% at 3 years, respectively. It is worth to stress that close monitoring of MRD (3–15 probes per patient) revealed different time adjusted dynamics in the molecular relapse incidence for bcr1/2 and bcr3 isoform. It was equal at each month during the whole period of follow-up (36 mo from treatment start) for bcr1/2 patients, and clustered in two periods (3–12 mo) and (20–36 mo) for bcr3 patients with 8 months of absence of positive signal. So we may suppose that each isoform of PML-RARa fusion gene determines its distinctive course of MRD while standard APL treatment. This data once again shows discrepancies in the biological features of APL with bcr1/2 and bcr3 isoforms.
Prognostic Significance of FLT3 Mutations in Pediatric Non-Promyelocytic Acute Myeloid Leukemia.
Authors: Kang Hyoung Jin , Hong Seung Hee , Kim In Ho , Park Byung-Kiu , Han Kyou Sup , Cho Han Ik , Shin Hee Young , Ahn Hyo Seop
Published: April 2020

Flt3 is a receptor tyrosine kinase involved in the survival of hematopoietic stem cells and activating mutations of Flt3 were reported to have prognostic significance in AML. This is the first study analyzed the Flt3 mutations in pediatric non-promyelocytic AML patients received same treatment scheme. Total 61 patients received BH-AC, idarubicin, 6-TG, and intrathecal Ara-C as an induction therapy. Once in complete remission (CR), patients with a matched related donor received BMT after two courses of consolidation therapy while those without donor received autologous peripheral blood stem cell transplantation with BCNU, etoposide, Ara-C, and cyclophosphamide conditioning regimen after four courses of consolidation therapy. Flt3 internal tandem duplication (Flt3/ITD) of juxtamembrane domain and D835 mutation (Flt3/D835) of tyrosine kinase domain were analyzed with PCR of genomic DNA. The incidence of Flt/ITD and Flt D835 were 6.6% (4/61) and 3.3% (2/61), respectively. After induction therapy, the CR was archived in 93.4% of the patients and all six patients with Flt3 mutations archived CR. The overall survival and event free survival of all patients were 58.8% and 45.6%, respectively. The disease free survival (DFS) was same as the relapse free survival (48.9%) because all events after CR were relapse. The patients with Flt3/D835 are still alive after autologous peripheral blood stem cell transplantation. The DFS of patients with Flt3/ITD (0%) was lower than that of others (51.9%) significantly (P = 0.0062). Flt3/ITD was the sole adverse prognostic factor for DFS in multivariate analysis (RR=5.6). The patients with Flt3/ITD relapsed very early even after receiving bone marrow transplantation from matched related donor with little BuCy conditioning. New therapeutic scheme such as stem cell transplantation with very intensive conditioning just after complete remission could be applied in pediatric non-promyelocytic AML patients with Flt3/ITD mutation.

Figure Figure
Predictive Value of Cytogenetics on Outcomes of Stem Cell Transplantation in Acute Myeloid Leukemia.
Authors: Park Jinny , Lee Mark H. , Lee Se-Hoon , Park Joon Oh , Kim Kihyun , Jung Chul W. , Kim Won Seok , Park Young Suk , Im Young-Hyuck , Kang Won Ki , Park Keunchil
Published: April 2020

Cytogenetic analysis performed at diagnosis is generally recognized as the single most valuable prognostic factor in AML. Several reports have suggested that cytogenetic characteristics present at diagnosis are associated not only with response to induction therapy for adult AML but also with the outcomes of postremission therapy. Our aim in this study was to examine the relationship of disease karyotypes at diagnosis with the outcomes of stem cell transplantation (SCT) as a postremission therapy in the first remission (CR1) of AML. To describe the differences and similarities used by different cooperative groups and to guide future investigations, the cytogenetic data presented in this study were coded and analyzed using two different classification schemes, SWOG and MRC. Forty-six AML patients younger than 56 years old who had undergone allogeneic or autologous SCT as postremission therapy were analyzed. Patients were categorized into favorable, intermediate, unfavorable, and unknown cytogenetic risk groups based on pretreatment karyotypes according to SWOG and MRC criteria. Prior to SCT, all patients received induction and consolidation chemotherapy consisting of idarubicin and cytarabine. Eight patients died during the median follow-up of 37.8 months (range, 7.6–91.8). The 3-year disease-free survival (DFS) and overall survival (OS) rates were 70.9% and 76.2% for all patients (n = 46). In univariate and multivariate analyses, cytogenetic risk status as grouped by the SWOG criteria was a significant prognostic factor for OS in patients with AML in CR1 who underwent SCT as postremission therapy (P = 0.017, P = 0.009). These data suggest that the pretreatment cytogenetic risk factor is the most valuable prognostic factor for OS in patients with AML in CR1 who undergo SCT as a postremission therapy. In particular, the SWOG criteria are superior in predicting OS and survival in our analysis. However, the unknown risk group in the SWOG criteria and the intermediate risk group in the MRC criteria are heterogeneous, and analyses that are more extensive are required. What is clearly needed is a generally accepted cytogenetic classification system and a leukemia cytogenetic database to provide prognostic scoring and real-time data updates, principally for the less frequent and secondary cytogenetic aberrations. Since the current analysis contains only data from patients who received SCT, this analysis has the possibility of being biased due to selection of patients with good performance and the exclusion of those with early relapses. However, the strengths of this trial included its well-defined eligibility criteria, including pretreatment cytogenetic studies, uniform treatment regimens for induction and postremission therapies, and the time between CR1 and SCT.
The Expression of Fas Antigen (CD 95) on Clonogenic Acute Myeloid Leukemia Cells Correlates with Their In Vitro Sensitivity to Farnesyltransferase Inhibitor (FTI; Zarnestra TM).
Authors: Szostek Marta , Zdzilowska Edyta , Skotnick Aleksander B.
Published: April 2020

The farnesyl transferase inhibitors (FTIs) have demonstrated significant antitumor activities in vitro and in vivo due to their ability to inhibit of Ras- oncogen farnesylation. Besides Ras, there are many other farnesylated proteins in cancer pathways which are potential targets for FTI. In this study, the in vitro effect of the FTI (Zarnestra, Z: provided kindly by Johnson & Johnson Pharmaceutical Research and Development) on clonogenic leukemia cells, expressing CD 34 and Fas were examined. All evaluated patients fullfilled the diagnostic criteria for high-risk AML and demonstrated the CFU-L in vitro growth, or/and expression of CD34 antigen on the myeloblast cells. Bone marrow mononuclear cells from 30 AML patients (M0=3, M1=15, M2=5, M3=1, M4=5, M5=1) were isolated and were plated in serum-free methylcellulose medium with or without growth factors (Methocult, Stem Cell Technologies, Vancouver, Canada) for the 14 days in vitro cultures. The expression of Fas (CD 95) on AML cells were also studied (anti-Fas APO-1, FITC- CD 95, Becton Dickinson, USA) by flow cytometric analysis using Cell Quest Software (Becton Dickinson, USA). MNC incubated with increasing concentrations of Z for 24, 48 and 72 days were analysed for apoptosis (staining for annexin-possitive cells, Annexin V Kit, Pharmingen, USA).

The percentage of Fas positivity was variable (range 20–94%) and was related to the morphologic FAB classification with the highest expression in AML-M1 patients (range 28–94, mean 72%, p<0.01).. No relationship was found between Fas and the CD 34 antigen expression. The in vitro CFU-L growth in the presence of cytokines was observed in 19/30 patients (63%) and 37% patients showed the spontaneous CFU-L or no colony growth of blast cells in the in vitro culture. In the majority of evaluated AML cases, a time and dose dependent proapoptotic in vitro effect of Z was found. The above effect was the most frequent in AML-M1 patients (11/23). Within Z concentration between 20–300 nM, the significant increase of apoptosis cell number (mean 60% AN+/PI− cells) was observed. After 24, 48 and 72 h, incubation with Z the number of patients whose cells underwent apoptosis were 49%, 13% and 8% respectively. The Z induced apoptosis showed significant correlation with Fas expression on AML cells.(P<0.01). The myeloblast apoptotic was not detected in the patients (40 %) demonstrated spontaneous CFU-L growth and the lack of Fas expression on blast cells (even after 120 h Z. exposure).

Our observations suggest that Z induced apoptosis of AML cells might be mediated at least in part by Fas, expressed on primitive, clonogenic leukemic progenitor cells. Induction of apoptosis throught the Fas pathway might be one of the new approach for effective leukemia therapy.
G Protein as Subunit T393C Polymorphism Is Associated with Progression-Free Survival in Chronic Lymphocytic Leukemia.
Authors: Nueckel Holger , Frey Ulrich H. , Duehrsen Ulrich , Siffert Winfried
Published: April 2020

Heterotrimeric G proteins mediate intracellular transduction of many hormone-receptor interactions. Activation of the G protein αs subunit (Gαs) upon stimulation of specific G Protein coupled receptors results in increased levels of the second messenger cAMP which has been shown to induce apoptosis in B-cells. On this background we investigated a potential association of a common silent single-nucleotid polymorphism (T393C) in the gene GNAS1 on chromosome 20 encoding Gαs with disease progression in patients with chronic lymphocytic leukemia (CLL).

We genotyped the DNA from 109 patients with CLL and 100 healthy controls using PCR amplification followed by restriction analysis. Comparing the CLL patients with healthy controls we found no significant differences in allele frequencies or genotype distributions (CLL patients: 393CC 34 vs CT 51 vs TT 24; healthy controls: 393CC 25 vs CT 50 vs TT 25). However, progression-free survival (PFS) was significantly shorter in 393CC patients compared to T393 allele carriers (median PFS CC: 36 months; T-allele: median PFS not reached; log rank test: p=0.029; adjusted for Binet stage) after 5 years observation time. In multivariate analysis the T393C polymorphism was an independent prognostic factor for PFS.

Our data suggest that the GNAS1 T393C polymorphism is not associated with the risk for B-CLL. However, the T393C polymorphism may be a prognostic marker for PFS. It is hypothesized that the 393T-allele is associated with increased cAMP concentrations and, therefore, with higher rates of apoptosis. Further studies assessing differences in the signaling pathway dependent on genotype are underway.
Prognostic Significance of FLT3 ITD and Asp835 Mutations in Patients with Acute Myeloid Leukemia (AML) - a Single Institution Experience.
Authors: Nahajevszky Sarolta , Andrikovics Hajnalka , Lovas Nora , Tordai Attila , Poros Anna
Published: April 2020

Treatment outcome in patients with AML is determined by a variety of prognostic factors such as WBC, LDH, age, presence of chromosomal aberration. Recently, two acquired genetic alterations, internal tandem duplications (ITD) and Asp835 codon mutations of the fms-like tyrosine kinase 3 (FLT3) gene were reported in AML as prognostic factors. 66 consecutive adult patients (39 females and 27 males) were treated with newly diagnosed AML in our institute between January 2002 and June 2004. The median age of onset was 49±15 (range 17–83) years. The gender distribution was 1.44 (female/male). We analyzed FLT3 ITD and Asp835 mutations at the time point of diagnosis by fluorescent PCR and PCR-RFLP methods. ITD was present in 22.7% (15/66) of the patients and Asp835 mutations were detected in 7.6 % (5/66). Two patients carried both mutations. 13 of 15 ITD-positive patients had myelomonocytic or monocytic leukemia (M4 and M5 in the FAB classification system). There was no difference between the numbers of ITD-positive and negative patients in complete remission (10/15 [66.7%] vs. 31/51 [60.8%]). In the ITD-positive patient group, the relapse rate was increased (5/15 [33.3%] vs. 7/51 [13.7%]) however, this difference was statistically not significant. The age of onset was greater than 40 years in all Asp835 mutation-positive patients and in this group, no relapse occurred within the follow-up time. The remission rate (4/5) of this group was high in spite of the old age of onset. The single Asp835-positive patient also carried the ITD mutation and showed primary resistance. Our results are in agreement and confirm earlier observations for the ITD mutation in a different patient population. In our relatively small patient group the Asp835 mutation seemed to have a beneficial effect.
Clinical Significance of AML/ETO Fusion Gene mRNA in Acute Myelogenous Leukemia.
Authors: Master Yungui Wang , Jin Jie , Chen Zhimei , Liang Yi
Published: April 2020

Acute myelogenous leukemia (AML) patients with normal-karyotype may have undetected chromosome aberrant that could affect prognosis. Screening for known AML-specific genetic abnormalities using the reverse transcription polymerase chain reaction (RT-PCR) may help in arriving at a more definitive prognosis. And RT-PCR is the most sensitive method to detect Minimal residual disease (MRD). Nested PCR was a frequently used method to amplify AML1-ETO fusion gene. To further comprehend the relationship between AML1-ETO mRNA expression in AML and its clinical significance in Eastern China. We investigated the prevalence of t(8;21) (q22;q22) and AML1/ETO fusion gene in 461 unselected de novo patients with AML by single RT-PCR and compared the results of cytogenetic analysis with these of RT-PCR. The patients’ median age was 29(1–76).

The results were used in diagnosis of 461 de novo leukemic patients, and 70 AML1-ETO-postive patients were followed up. 107 in 461 patients (23.2%) AML1/ETO was detected by RT-PCR. 98 in 461 patients (21.3%) showed t(8;21) (q22;q22) in karyotype analysis. All patients who had t(8;21) (q22;q22) in conventional karyotyping also showed the gene rearrangement in molecular analysis. The results showed that AML1/ETO mRNA could be expressed in cells from AML-M1, AML-M2 and AML-M4 patients. The complete remission rate in AML1/ETO-positive patients was significantly higher than that in AML1-ETO-negative patients (without M3 subtype patients)[80.4% (86/107) vs 70.6% (191/269)]. The AML1-ETO-postive patients who became negative after chemotherapy had an optimistic outcome, but the patients who demonstrated persistence of AML1-ETO mRNA had a poor prognosis. In chemotherapeutic group, patients whose AML1/ETO expression turning from negative (3 cases) or faint positive (1 case) to positive relapsed later. So PCR becoming positive again indicated relapsing. Only 7 was detected in 48 M2 t(8:21) patients who had been maintaining remission for more than 18 months.

RT-PCR detected the overall AML1-ETO-positive rate in AML was from 6% to 13% in Western country. But our results showed the rate is 23.2%. One reason might be that the patients involved in our observation were younger than those patients involved in other observation group reported.. The other reason, which might be more important, was that the differences between China and Western country in race and region.Our results showed single RT-PCR method was sensitive enough to detect AML1-ETO mRNA. These observations suggest that AML1/ETO mRNA could disappear after chemotherapy or bone marrow transplantation. The patients had a great probability to relapse if the results of RT-PCR are continuously positive or change from negative to positive. So Regular detection is necessary for leukemia patients. We can monitor the minimal residual disease by detecting AML1-ETO mRNA regularly to direct clinical therapy
The Prognostic Importance of Pre and Post Dendritic Cells in Autologous Transplantation for Diffuse Large B Cell Lymphoma.
Authors: Bolwell Brian J. , Pohlman Brad , Andresen Steve , Serafino Sheila , Sobecks Ronald , Kuczkowski Elizabeth , Curtis Julie , Rybicki Lisa , Maciejewski Jarek , Baker Janet , Cherni Kelly , Bernhard Laura , Kalaycio Matt , Hsi Eric , Theil Karl
Published: April 2020

Pre and post transplant dendritic cell (DC) levels may correlate with survival, GVHD, and relapse in allogeneic BMT. Whether they have prognostic importance in autologous peripheral blood progenitor cell (PBPC) is unknown. We prospectively collected pre and post dendritic cell levels, including DC1 and DC2 levels in 53 patients with diffuse large B cell lymphoma (DLBCL) to investigate their potential prognostic importance. Pre-transplant analysis of DC1 and DC2 by flow cytometry was obtained 24 hours prior to the initiation of VP-16 (2 gm/m2) or G-CSF (10 mcg/kg/d) (n=4) for PBPC mobilization; post transplant samples were obtained 6 weeks after transplant. 51 of 53 (96%) were sensitive to chemotherapy at the time of transplant; 34% received prior radiation therapy; 15% had an elevated LDH at transplant. 49 of 53 received VP-16 + G-CSF for stem cell mobilization and 4 patients received G-CSF alone. 98% received a preparative regimen of Bu/Cy/VP and one patient received Busulfan alone. In a univariate analysis, higher numbers of pre-transplant DC1 cells and pre-transplant total DC (DC1 + DC2) cells correlated with improved survival, as shown graphically below:

Figure Figure

Patients received a median CD34+ cell dose of 8.7 x 106/kg. DC values did not correlate with CD34+ cell yield. Post-transplant DC1 (p=0.03), DC2 (p=0.035), and post-transplant total DCs (p=0.04) also correlated with improved survival. In all cases, more DCs was associated with a better outcome. In a multivariable model, pre-transplant total DC number (p=0.028), and a normal LDH at the time of transplant (p=0.017) correlated with improved outcome. Median follow up of surviving patients is 15 months. Of 26 patients with pre-transplant total DC numbers greater than 9.10 x 106/mL, 77% of patients are alive, as compared to 56% of patients with pre-transplant total DC numbers less than 9.10 x 106/mL (n= 27)(p=0.022). The high DC group had fewer relapses and fewer late infections. We conclude that higher pre-transplant total DC numbers are associated with improved outcome in ABMT for DLBCL.
Growth Hormone Deficiency after Chemotherapy for Acute Lymphoblastic Leukemia in Children Who Have Not Received Cranial Radiation.
Authors: Haddy Theresa B. , Nunez Susan B. , Mosher Revonda B. , Reaman Gregory H.
Published: April 2020

Chemotherapy-related growth failure is a significant problem in children with acute lymphoblastic leukemia (ALL). Growth impairment after cranial radiation (CR) is likely to result in diminished adult height, but growth impairment following chemotherapy without CR is usually followed by catch-up growth and normal final height. We report two ALL survivors treated with chemotherapy without CR who required growth hormone (GH) replacement due to absence of catch-up growth. A 15 year old boy and a 12 year old girl, off therapy for 9 and 6 years, respectively, developed growth failure. Peak GH responses to stimulation using arginine and clonidine were inadequate, 3.4 ng/ml and 0.1–3.0 ng/ml, respectively (normal >10 ng/ml). Additional studies included bone age, magnetic resonance imaging of the hypothalamic-pituitary area, insulin-like growth factor (IGF-1), insulin-like growth factor-binding protein (IGF-BP3), and thyroid function tests. Other causes of growth failure were ruled out; the children had received neither CR nor radiation of the long bones, were not subjected to transplantation, and showed no evidence of premature puberty or hypothyroidism. Chemotherapy included methotrexate, 6 mercaptopurine, vincristine, adriamycin, cyclophosphamide, l-asparaginase, dexamethasone, cytarabine, 6 thioguanine, and intrathecal methotrexate. Both children responded to GH replacement therapy. Of 235 ALL survivors treated at our single institution since 1997 with chemotherapy without CR, the 2 we report here were diagnosed with GH deficiency resulting in growth deceleration, generally thought to be primarily a complication of CR. Children treated for ALL should be carefully monitored for growth failure.
CD133+ Hematopoietic Cells Successfully Reconstitute Hematopoiesis Following Autologous Peripheral Blood Stem Cell Transplantation.
Authors: Hale Gregory A. , Horwitz Edwin , Leung Wing , Woodard Paul , Eldridge Paul , Yusuf Usman , Benaim Ely , Kasow Kimberly , Srivastava Kumar , Handgretinger Rupert
Published: April 2020

CD133 is a unique antigen found on hematopoietic precursor cells, with a limited expression on non-hematopoietic cells. These features make it an attractive marker for obtaining tumor-free hematopoietic grafts. We conducted a prospective clinical trial for patients with solid tumors and lymphomas who required autologous HSCT. 11 children (6 male, 5 female) had CD133+ peripheral blood stem cells (PBSC) collected for subsequent autologous HSCT. The median age was 12.4 yrs (range, 2.2–26). Diagnosis included Hodgkin lymphoma (1 stable disease, 2 PR), neuroblastoma (2 PR), NHL (2PR), Ewing sarcoma (1 stable disease), CNS PNET (1 PR), and desmoplastic small round cell tumor (1 PR). All had received priming chemotherapy with growth factor support. PBSC were collected in 1 or 2 procedures when the absolute CD34+ count was ≥ 40/ul. A stem cell product was cryopreserved as a backup. The PBSC product was processed on the CliniMACS device with positive selection methodology using the CD133 antigen. Prior to CD133 selection, the graft contained 0.3–4.9% CD34+ cells and 0.3–4.4% CD133+ cells. Following CD133 selection, the graft contained 45.1–98.4% CD34+ cells and 45.9–98.8% CD133+ cells. The stem cell products contained a median of 5.4 x 106 CD34+/kg (range, 2.61–7.89) and 5.3 x 106 CD133+/kg (range, 2.54–8.04). One patient who had PBSC collected has not yet proceeded to HSCT. All were conditioned with busulfan 37.5 mg/m2/dose for 16 doses and melphalan 70 mg/m2 for 2 doses. A cell dose from 2 x 106 CD133+/kg to a maximum of 8 X 106 CD133+/kg was infused. G-CSF 5 mcg/kg/day was initiated on day +5 and continued until ANC ≥ 3,000/mm3 for 2 consecutive days. All 10 patients engrafted and no patient required infusion of the back-up stem cell product. No infusion reactions were observed during infusion of the stem cell product. The median time to ANC ≥ 500/mm3 was day + 11 (range, 10–13) for all 10 patients. Of the 8 evaluable patients (1 had hemorrhagic cystitis, 1 severe epistaxis), all achieved an unsupported platelet count of greater than 20,000/mm3 at a median of day + 17 (range, 12–20). This trial demonstrates that infusion of CD133+ hematopoietic cells can reconstitute hematopoiesis following myeloablative HSCT. Further studies are needed to describe the ability of CD133 selection to obtain tumor free hematopoietic grafts.
ZAP-70 Expression in Human B cells: Analysis of Normal Cells and Acute Lymphoblastic Leukemia (ALL) Cells with Pro/Pre B Phenotype.
Authors: Crespo Marta , Villamor Neus , Gine Eva , Colomer Dolors , Marafioti Teresa , Campo Elies , Montserrat Emili , Bosch Francesc
Published: April 2020

ZAP-70 is a protein tyrosine kinase of the Syk/ZAP-70 family that plays a critical role in the signal transduction from the T-cell receptor. In human lymphocytes, ZAP-70 gene has been reported to be expressed in T and NK derived cells, and in IgVH unmutated B-chronic lymphocytic leukemia cells. More recently, ZAP-70 expression has been shown to be required for the development of pro-B cells to pre-B cells in mice. To ascertain the expression of ZAP-70 gene in human immature B-cell stages, we analyzed ZAP-70 protein and/or mRNA in normal human B cells at different stages of B cell maturation, including pro/pre-B cells and tumoral cells from 20 B-ALL. ZAP-70 expression was assessed by flow cytometry (FC), immunofluorescence (IF), and/or by quantitative real time RT-PCR (QRT-PCR). In normal bone marrow, ZAP-70 expression was found only in T and in immature B cells (CD19+/CD10+/CD20 −). Moreover, T cells -but no mature B cells- from normal tonsil expressed ZAP-70, as assessed by QRT-PCR and IF. In B-ALLs, a high ZAP-70 expression by FC was observed in 9/13 cases (mean, 82.6%, range 60–99%), whereas in 4 cases ZAP-70 was barely detectable (mean, 13%). By QRT-PCR, 10/16 B-ALLs showed levels of expression similar to ZAP-70 non-expressing cell lines and normal B-cells, whereas in the remaining cases ZAP-70 expression was 3–4 times higher than in normal mature B-cells. Taken together, a high expression of ZAP-70 was found in 11/21 (52%) B-ALLs. No relationship was observed between the level of ZAP-70 expression and the B-ALL maturation status. In conclusion, among normal B cell subsets ZAP-70 expression is restricted to B-cells with pro/pre phenotype. In addition, ZAP-70 is expressed in 52% of B-ALLs, probably as a reflection of their B-cell origin.
Higher Expression of Cytochrome-c and Caspase-3 in the Primary Acute Lymphoblastic Leukemic Cells with Negative Expression of VEGF and Its Receptors Than with Positive Expressed Cells under VP16 Induction.
Authors: Liang Aibin , Gu Long-Jun
Published: April 2020

Flow cytometry and immunofluorescent staining were employed to examine the apoptosis signals, cytochrome-c and Caspase-3, in the primary leukemia cells with or without expression of VEGF and its receptors and normal healthy donor’s lymphocytes. We found that primary leukemic cells and normal lymphocytes didn’t secrete Cyt-c from mitochondria and no caspase-3 activation without the treatment of VP16. Instead, after incubation with VP16 (30mg/L) for 8 hours, normal lymphocytes and most of de novo leukemia cells secreted Cyt-c into cytosol from mitochondria and had the activation of caspase-3 in the cellular plasma. However, primary leukemia blasts with higher expression of VEGF and its receptors revealed lower secretion of Cyt-C and down-regulated activation of Caspase-3. Results showed that Cyt-c secretion and Caspase-3 activation were prone to occur in the VEGF negatively expressed blasts than positively expressed cells. This indicates that lower secretion of Cyt-c from mitochondria and inactivation of Caspase-3 are two of the pivotal mechanisms of anti-apoptosis for refractory leukemia clone with highly expressed VEGF and its receptors.
A Risk Model for Early Intracranial Hemorrhage in Acute Leukemia.
Authors: Kim Hawk , Choi Seong-Jun , Lee Je-Hwan , Lee Jung-Shin , Lee Kyoo Hyung
Published: April 2020

Early intracranial hemorrhage (EICH), which is defined by noticeable ICH within 10 days of diagnosis, is a life-threatening hemorrhagic complication in patients with acute leukemia. To ascertain risk factors associated with EICH, we retrospectively analyzed 792 newly-diagnosed acute leukemia patients treated between July 1988 and March 2003. Thirty-one patients (3.9 %) had analyzable EICH. Multivariate analysis showed that female gender (OR = 3.064, P < 0.001), acute promyelocytic leukemia (OR = 8.797, P = 0.003), leukocytosis (OR = 6.056, P = 0.004), and prolonged prothrombin time (OR = 10.026, P = 0.016) were factors significantly associated with occurrence of EICH. Risk scores (RS) were calculated by a product of odds ratio and each risk factor (RF) and taking their sum, generating an RS ranging 0 to 27.943. The cutoff of RS 9 was of statistical significance to predict probability of EICH. Receiver-operating characteristics curve shows the sensitivity and 1 - specificity relative to risk score (AUC = 0.917; S.E. = 0.021; 95% CI, 0.876–0.958; Figure 1). In this regards, RF for EICH was classified as major (prolonged prothrombin time) and minor (female gender, acute promyelocytic leukemia, leukocytosis). Risk model demonstrated that EICH was low probable when no major RF and less than two minor RFs were present. Risk model for ICH in acute leukemia classified acute leukemia patients into two risk groups; low probable EICH group (LPG) and probable EICH group (PG). When applied to our patients, PG was positively correlated with more incidence of FICH than LPG (n = 27/173 vs. 4/619, P < 0.001). Induction chemotherapy could be undertook more frequently in LPG than in PG (p = 0.002, Table 1). Kaplan-Meier curves show the probability of EICH-free survival relative to probability of EICH. ICH-free survival was significantly longer in LPG than in PG (p < 0.0001, Figure 2). Our findings suggest that our risk model may predict the occurrence of EICH in patients with acute leukemia.

Table 1. The relationship of risk model and frequency of early intracranial hemorrhage (EICH) or performance of induction chemotherapy.

Figure Figure Figure Figure Low probable EICH group, n (%) Probable EICH group, n (%) P EICH (−) 615 (77.7 %) 146 (18.4 %) < 0.001 EICH (+) 4 (0.5 %) 27 (3.4 %) Induction chemotherapy (+) 563 (71.1 %) 143 (18.1 %) 0.002 Induction chemotherapy (−) 56 (7.1 %) 30 (3.8 %)
Characterization and Functional Analysis of a Murine Model for Chronic Myeloid Leukemia Stem Cells.
Authors: Sozer Selcuk , Echlin Debbie R. , Rossi Randall M. , Grimes Barry A. , Jordan Craig T.
Published: April 2020

The purpose of this study was to characterize the phenotype and analyze the functional characteristics of CML stem cells (CML-SCs) in a mouse model system. A retrovirus transduction model was employed using a vector expressing p210/BCR/ABL and the green fluorescent protein (GFP). Transplantation studies of BCR/ABL infected cells in recipient mice demonstrated an aggressive myeloproliferative disease within approximately 3 weeks, as has previously been observed with this approach. Phenotypic analysis of bone marrow from BCR/ABL mice showed contribution by the transduced cells to multiple lineages, suggesting a primitive stem/progenitor cell gives rise to the disease. Subsequent analysis of primary cells that arise in vivo showed the leukemia-initiating activity resides in cells that are Sca-1+/c-kit+/Lin-. Cell cycle analyses (as detected by BrdU incorporation) of the Lin−/c-kit+/Sca-1+ compartment of BCR/ABL mice demonstrated an enhanced cycle rate (approximately 2-fold) in the leukemic population, signifying a possible increase in the self-renewal or proliferative properties of this primitive compartment that could lead to development/progression of the disease. Further, comparison of the BCR/ABL mice and GFP control mice revealed preferential over-expression of CD24 (heat-stable antigen) in the primitive leukemic cells (90% vs. 52% in normal counterparts) demonstrating a possible BCR/ABL induced activation of the antigen. Sorting and serial transplantation studies of CD24+ vs CD24− cells from BCR/ABL mice showed that the leukemia-initiating activity resides in the CD24+/Lin− population. Collectively, these data indicate that BCR/ABL expression increases proliferation of primitive CML cells and up-regulates the CD24 antigen. We suggest that the CD24 antigen represents a potential marker to distinguish normal from CML stem cells and that it may confer specific functional properties on the CML population.
Comparison of Platelet-Rich Plasma Serotonin Level and Quantitative Neutrophil PRV-1 Assay in Chronic Myeloproliferative Disorders.
Authors: Koch Cody A. , Lasho Terra L. , Tefferi Ayalew
Published: April 2020

In a prospective study of 100 subjects, an ELISA assay was used to measure platelet-rich plasma (PRP) serotonin levels in patients with polycythemia vera (PV; n=26), essential thrombocythemia (ET; n=14), myelofibrosis with myeloid metaplasia (MMM; n=26), secondary or spurious polycythemia (SP; n=20) and controls (n=14). Neutrophil PRV-1 expression was concurrently assayed by real-time PCR in 69 patients (23 PV, 17 SP, 12 ET, 7 MMM, 10 controls). Table 1 outlines the median and range of measured values across the different study groups.

Table 1

Controls SP PV ET MMM Age in years N/A 49 (18–77) 64 (32–83) 52 (18–81) 60 (22–80) Plt count x 109/L N/A 249 (157–371) 509 (191–940) 613 (284–1100) 259 (64–955) PRP serotonin; ng/109 plts 567 (360–1071) 609 (369–1780) 205 (0–496) 385 (137–1026) 90 (0–278) PRV-1/GAPDH ratio 1.28 (1.20–1.43) 1.23 (1.13–1.38) 1.03 (0.84–1.28) 1.24 (0.99–1.44) 1.25 (1.11–1.40)

A highly significant difference (p < 0.0001), in PRP serotonin levels, was seen among the different disease categories and control group (table 1). The lowest levels were registered for MMM and PV with a statistically significant difference between the two groups (p = 0.015) but a large degree of overlap. The results in both were significantly as well as markedly lower than those of controls as well as SP with only slight overlap in the PV group and no overlap in the MMM group (p < 0.0001; table 1). The PRP serotonin levels in patients with ET were significantly reduced compared to both controls (p = 0.019) and SP (p = 0.002) and significantly increased compared to both PV (p = 0.0008) and MMM (p < 0.0001). However, there was a substantial degree of overlap in values between ET and other disease categories and controls. Neither aspirin nor cytoreductive therapy correlated with PRP serotonin levels (p = 0.116 and p = 0.148, respectively). Similarly, within a specific disease category, PRP serotonin level did not significantly correlate with platelet count, hemoglobin level, leukocyte count, spleen size, disease duration, or gender. However, an inverse correlation with age was noted only in MMM. In general, there was not a significant correlation between PRP serotonin level and neutrophil PRV-1 expression in each of the three myeloproliferative disease variants. Tests of sensitivity, specificity, and accuracy in distinguishing PV from SP were 92, 95, and 93% for the PRP serotonin assay and 78, 95, 86% for neutrophil PRV-1 assay, respectively. The current study suggests that PRP serotonin concentration might be considered as one of several biological markers that complement each other for the diagnosis of PV.
Abnormal Expression of the Plasma Cell Differentiation Factor X-Box Protein 1 (Xbp-1) in Waldenstrom’s Macroglobulinemia.
Authors: Xu Lian , Leleu Xavier , Hunter Zachary R. , Santos Daniel D. , Ho Allen W. , Hatjiharissi Evdoxia , O’Connor Kelly , Manning Robert J. , Chemaly Mariana , Branagan Andrew R. , Patterson Christopher J. , Verselis Sigitas , Fox Edward , Anderson Kenneth C. , Treon Steven
Published: April 2020

INTRODUCTION: Xbp-1 is a ubiquitously expressed basic leucine zipper protein which serves as key transcription factor for plasma cell differentiation and immunoglobulin production. XBP-1 mRNA undergoes splicing at an IRE-1a specific cleavage site to produce a spliced form (Xbp-1s) which due to a frame shift results in a more stable and potent transcription factor than the unspliced form (Xbp-1us). Previous observations demonstrated that Xbp-1 was highly expressed in multiple myeloma (MM), though its expression in other B-cell disorders including Waldenstrom’s macroglobulinemia (WM) remains to be clarified.

METHODOLOGY: In this study, we investigated the different forms of Xbp-1 in CD19+ selected bone marrow (BM) cells from 61 patients with the consensus panel definition of WM using semi-quantitative PCR analysis. CD19+ selected BM cells from 6 healthy donors, and CD138+ selected BM cells from 4 MM patients were used as controls. All RT-PCR results were normalized to b-actin levels. Sequencing of Xbp-1 in CD19+ selected LPC was also performed for 20 WM patients.

RESULTS: In 9/61 (15%) WM patients, Xbp-1 transcripts were undetectable. Among WM patients expressing Xbp-1, as well as all MM patients evaluated, higher levels of XBP-1us were observed in comparison to normal healthy donors (p=0.001). Decreased XBP-1s transcript levels were also observed among WM and MM patients versus healthy donors, but did not reach statistical significance. Sequence analysis of Xbp1 in CD19+ selected BM LPC demonstrated variants 10/20 WM patients in exon 1 that are currently under investigation.

CONCLUSIONS: Abnormal expression of Xbp-1 is common in patients with WM, and includes loss of expression, and aberrations in splice patterns. The genetic basis for these findings is under investigation.
Identification of Surrogate Prognostic Markers and Genes Implicated in Immunoglobulin Gene (IgVH) Somatic Hypermutation in Small B-Cell Lymphomas.
Authors: Tracey Lorraine , Aggarwal Mohit , Algara Patrocinio , Garcia Juan F. , Garcia-Cosio Monica , Rodriguez Antonia , Martinez Nerea , Camacho Francisca I. , Roncador Giovanna , Sanchez-Aguilera Abel , Ruiz-Ballesteros Elena , Mollejo Manuela , Garcia-Marco Jose A. , Piris Miguel A.
Published: April 2020

Immunoglobulin (Ig) gene somatic hypermutation (SHM) is a prognostic factor in small B-cell lymphomas, as demonstrated by studies reporting that hypermutated chronic lymphocytic leukemia (CLL) and splenic marginal zone lymphoma (SMZL) cases show a better prognosis, while hypermutated mantle cell lymphoma (MCL) cases display specific clinicopathological features (leukemic course, increased survival).

However both the mechanisms and markers of SHM are poorly characterized, with the partial exception of the role of the genes ZAP70 and AID. With the purpose of identifying SHM surrogate markers in small B-cell lymphomas, we analyzed IgVH mutational status and expression profiles of 93 small B-cell lymphoma patient samples including SMZL (24 cases), MCL (33 cases) and CLL (36 cases). Patients were classified into two groups: high SHM (>5% mutations) and low SHM (<5% mutations). T-test analysis with 100,000 permutations was performed and 39 genes were identified whose expression is significantly different (p<0.005, FDR<0.05) between cases with high and low SHM burdens.

To dissect the molecular mechanisms of Ig somatic hypermutation and validate the observed findings, SHM was induced in a model system and compared to results in patient samples. The BL2 cell line was used, and SHM was induced by treating the cells with IL4 and CD40. After 24 hours, active SHM was confirmed by western blot using an anti-AID monoclonal antibody on treated and untreated cells. These same cells were analyzed using microarrays, to identify the genes that were induced or downregulated during the SHM activation. A total of 29 genes (18 upregulated and 11 downregulated) were identified which are significantly differentially expressed in the cell line model during induction of SHM (expression change >0.4 Log2 scale) and are also differentially expressed between cases with high and low SHM (p<0.001, FDR<0.1). In both analyses, the upregulated genes are implicated in transcription, DNA repair and replication and chromosome maintenance, correlating well with previous hypotheses indicating that active transcription is necessary for SHM. Based on these observations, a group of 10 key genes, implicated in DNA repair, replication and transcription, were selected and protein expression was analyzed in a set of tissue microrrays containing 150 paraffin embedded small B-cell lymphoma cases, for which clinical data is available and SHM status is known. These proteins may provide a surrogate marker for prognosis and/or analysis of IgVH SHM in patient samples using paraffin-embedded tissue samples.
A Novel Mutation in the von Willebrand Factor Propeptide Causes Defective Regulated Storage in Platelets and Weibel-Palade Bodies.
Authors: Haberichter Sandra L. , Budde Ulrich , Drewke Elke , Obser Tobias , Wermes Cornelia , Schneppenheim Reinhard
Published: April 2020

Two children from a Turkish consanguineous family suffering from significant mucocutaneous bleeding episodes were diagnosed with von Willebrand disease type 2A. The relative reduction of large plasma von Willebrand factor (VWF) multimers together with the absent triplet structure was consistent with type 2A (phenotype IIC) VWD. Surprisingly, platelet VWF was completely deficient of multimers beyond the VWF protomer, suggesting defective α-granular storage of larger multimers. The two patients were nearly unresponsive to DDAVP, consistent with a lack of regulated VWF release from endothelial cell Weibel-Palade bodies, suggesting defective storage in endothelial cells as well as in platelets. We identified the homozygous mutation in these patients, N528S, in the D2 domain of the VWF propeptide (VWFpp). To examine the effect of the N528S mutation on VWF regulated storage, a VWF expression vector containing the mutation was constructed and expressed in AtT-20 cells that contain an intact regulated storage pathway. Transfected cells were immunofluorescently labeled and the intracellular localization of VWFpp and VWF were examined by confocal microscopy. In cells expressing N528S-VWF, VWF was not stored in granules but was localized to the endoplasmic reticulum (ER). Our previous studies have shown that VWF granular storage is initiated by VWFpp: VWF is trafficked to storage granules through its non-covalent association with VWFpp. To determine if the loss of granular storage of N528S-VWF was due to defective VWFpp trafficking, an N528S-VWFpp expression vector was constructed and expressed in AtT-20 cells. The N528S-VWFpp was found to be colocalized with endogenous ACTH-containing storage granules, demonstrating normal granular trafficking of the variant VWFpp. The N528S-VWFpp was co-expressed with propeptide-deleted, mature VWF (Δpro) to examine regulated storage when the two proteins are expressed in trans. In the majority of transfected AtT-20 cells, N528S-VWFpp was trafficked to granules while the coexpressed Δpro appeared to be ER-localized, consistent with a loss of non-covalent association between VWFpp and VWF. In a subset of cells neither VWFpp nor VWF were stored in granules. In contrast, when N528S-VWF was expressed as a full-length construct (in cis), VWFpp granular storage was observed only in a subset of cells and the majority of transfected cells demonstrated ER-localization of both proteins, suggesting the loss of regulated storage of N528S-VWF is not due to defective VWFpp sorting, but rather an aberrant interaction of VWFpp with the mature VWF protein. Together these data indicate that the phenotype observed in patients with N528S variant VWF is result of defective multimerization, storage, and secretion due to an aberrant VWFpp/VWF interaction.
The Role of Lipid Rafts in the Assembly and Down-Regulation of the Factor X Activating Complex on the Activated Platelet Membrane.
Authors: Ahmad Syed S. , Su Ya-Chi , Walsh Peter N.
Published: April 2020

In a recent study of the role of detergent-insoluble platelet membrane microdomains (lipid rafts) in the assembly of the factor X (FX) activating complex, we have shown, contrary to expectations, that the formation of lipid rafts after incubation of platelets with the thrombin receptor activation peptide (SFLLRN, 25 μM, for 30 min) results in the down-regulation, rather than assembly, of the enzyme-cofactor-substrate complex by sequestering FVIIIa and FX in rafts and separating them from FIXa, which is excluded from raft fractions isolated by sucrose density gradient centrifugation of triton X-100 (0.25%) solubilized platelets. Since the FX-activating complex is assembled rapidly on platelets after incubation with low concentrations of either thrombin (>1 nM) or SFLLRN (25 μM), we have now examined the kinetics of FX-activation complex assembly at early time points after exposure of platelets to agonists. Washed and gel-filtered human platelets, activated with SFLLRN (25 μM) in the presence of FVIIIa (5 U/ml, 1.5 nM) and FX (125 nM) rapidly (within 2 min) developed the capacity to support maximal rates of FIXa (1 nM) catalyzed FX activation that was transient and decayed to baseline within 5 min after exposure to agonist. At these early time points (0.5, 1 and 2 min), platelets activated with SFLLRN (25 μM) in the presence of 125I-labled FVIIIa (nM), FIXa (nM) or FX (nM) and analyzed by sucrose density gradient centrifugation after solubilization in triton X-100 (0.25%) were shown to sequester within the raft fractions ~15% of FIXa, ~15% of FVIIIa and ~15% of FX, whereas at later time points (5–30 min) only FVIIIa (~25%) and FX (~45%) were localized in rafts, from which FIXa was completely excluded. These results strongly suggest that platelet membrane microdomains (lipid rafts) form rapidly after exposure of platelets to PAR-1 agonists to colocalize the enzyme-cofactor-substrate complex, which is transient since at later time points FIXa dissociates from rafts that sequester the FVIIIa-FX complex to down-regulate FX activation.
Heparin Chain Length Dependence of Factor Xa Inhibition by Antithrombin in Plasma.
Authors: Rezaie Alireza R.
Published: April 2020

Heparin anticoagulants function by enhancing the inhibition of coagulation proteases by the serpin antithrombin (AT). Evaluation of the specific anti-factor Xa (fXa) activity of therapeutic heparins in the physiologically relevant plasma-based clotting assays has not been feasible since thrombin, the final protease of the cascade, is the primary target for inhibition by AT in the presence of heparin. To circumvent this problem, we developed an assay in which the native AT in plasma was replaced with an AT mutant which exhibits identical affinity for heparin and near normal reactivity for fXa, but does not react with thrombin and other coagulation proteases in either the absence or presence of heparin. This assay was used to distinguish the anti-fXa activity of different molecular weight heparins from their anti-thrombin activity in clotting assays which were initiated by triggers of either extrinsic or intrinsic pathways of coagulation. The results suggest that the acceleration of fXa inhibition by AT exhibits a marked heparin chain-length dependence, with fondaparinux (a pentasaccharide) having the lowest and unfractionated heparin having the highest effect. Interestingly, comparative studies revealed that the fondaparinux-catalyzed acceleration of thrombin inhibition by AT also makes a significant contribution to prolongation of the clotting time, possibly suggesting that the anticoagulant function of the therapeutic pentasaccharide is mediated though the inhibition of both fXa and thrombin.