bluuebunny/crossref_metadata_2025 huggingface dataset:
GM-CSF in Stem Cell Transplant (SCT) Recipients: Impact of Graft-Versus Host Disease on Invasive Fungal Infections (IFIs)
Authors: Safdar Amar , Rodriguez Gilhen , Georgescu Georgina , Champlin Richard
Published: April 2020

Background: GM-CSF is thought to be beneficial in SCT patients with IFI. The impact of GM-CSF in SCT recipients with GVHD and IFI was evaluated

Methods: A retrospective case-matched (GM-CSF [n = 12] vs. no GM-CSF [n = 27]) study was undertaken to compare outcomes if IFIs diagnosed >100 days following transplantation during 2000–2006. A response was defined as complete or partial response (CR/PR) and considered 3 months after IFI diagnosis or earlier in patients with earlier response. All value are given as median ± s.d. The systemic antifungal therapy in > 60% of patients included echinocandin and anti-mold triazole, or polyene agent.

Results: Please refer to the table below. Patients who received GM-CSF were younger (age 36 ± 17 vs. 51 ± 13 years); neutropenic (<500 cells/uL) at onset of infection (50% vs. 11%; P = 0.014); had lower monocyte counts (10 ± 236 vs. 220 ± 575 cells/uL; P = 0.021) and frequently required intensive care unit stay (50% vs 15%; P = 0.043). A 50% response was observed in the GM-CSF treated SCT recipients with GVHD and IFIs, whereas, 41% response occurred in the group who received no concurrent Gm-CSF (P = 0.6). Similarly, IFI-associated deaths were less frequently observed in GM-CSF treated patients (41%) compared with 55% seen in patients who had not received GM-CSF (P = 0.7). There was a slight increase in survival among SCT recipients treated with GM-CSF (48 ± 21 days vs. 34 ± 19 days in no GM-CSF group P = 0.05)

Conclusions: Late fungal infections in SCT recipients with GVHD, when treated with GM-CSF-based therapy had comparable outcomes despite having significantly more neutropenia, severe monocytopenia and ICU stay.

Characteristics GM-CSF N = 12 (5% No GM-CSF N = 27 (%) p-value Underlying Disease-Leukemia Relapsed/refractory Cancer 10 (83) 5 (42) 18 (67) 6 (22) 0.12 0.26 Proven and probable IFI 9 (75) 21 (78) 1 GVHD (extensive) 5 (42) 14 (52) 0.57 High Dose Steroids 7 (58) 18 (67) 0.72 Disseminated IFI 3 (25) 4 (15) 0.65 Breakthrough IFI 10 (83) 25 (93) 0.57 APACHI II Score at time of IFI diagnosis 11+/−4 (range 5–18) 12+/−3 (range 5–19) 0.47
CD3−cd(16+56)+ Cell Dose Is An Important Factor Determining Clinical Outcomes in Patients Undergoing Unmanipulated HLA-Identical Sibling Hematopoietic Stem Cell Transplantation
Authors: Tang Xiaowen , Peng Biyun , Zhu Mingqing , Depei Wu , Sun Aining , Ruan Changgeng
Published: April 2020

Background: It has been suggested that the dose of the cellular components in the graft may influence the outcome of transplantation, especially CD3 |CD(16+56)+ cell(NK cell) is believed to play an important role in improving the transplant outcome in terms of preventing graft-versus-host disease(GVHD) and graft rejection, and as a mediator of the graft-versus-leukemia(GVL) effect. The current study attempted to evaluate the role of CD3−CD(16+56)+ cell dose on the outcome of HLA-identical sibling hematopoietic stem cell transplantation.

Methods: We retrospective analyzed the outcomes of 72 patients receiving HLA-identical sibling hematopoietic stem cell transplantation between July 2005 and May 2008 in the first Affiliated Hospital of Soochow University. Diagnoses of these patients included acute leukemia(AL)(n=34), chronic myeloid leukemia(CML) (n=29), aplastic anemia(AA) (n=4), malignant lymphoma (n=3) and multiple myeloma (n=2). The median follow-up time is 19 months (range 0.5–36 months) after transplantation. Transplanted doses of CD3 |CD(16+56)+ cell were analyze in relation to engraftment, GVHD, infectious events, relapse and survival.

Result: The median dose of NK cell in bone marrow grafts from patients receiving G-CSF-mobilized was 0.91× 107/Kg. Therefore the group received a dose of CD3 |CD(16+56)+ cell above 0.91×107/Kg wass a higher group. There were no significant difference between two groups for neutrophil and platelet recovery. The incidence of II°-‡W°aGVHD in low group was much more than that of high group(41%VS 18.2%, P<0.05). and there was a significantly higher incidence of infection in patients receiving a lower dose of CD3 |CD(16+56)+ cell (46.1%VS 21.2%, P<0.05). However there was no significant association between CD3-CD(16+56)+ cell and either cGVHD or relapse.

Conclusions: High dose of CD3−CD(16+56)+ cell may play an important role in improving the outcome of transplantation in terms of reducing aGVHD and infection.
Allogeneic Stem Cell Transplantation for Core Binding Factor Alpha-Positive Adult Acute Myelogenous Leukemia with a Reduced Intensity Conditioning Regimen Consisting of Intravenous Busulfan, Fludarabine, and 400 cGy Total Body Irradiation
Authors: Kim Heeje , Min Woo-Sung , Cho Byung-Sik , Kim Yoo-Jin , Lee Seok , Min Chang-Ki , Cho Seok-Goo , Lee Jong-Wook , Kim Chun-Choo
Published: April 2020

Translocation (8;21) in core binding factor alpha acute myelogenous leukemia (CBFA-AML) leads to the generation of the AML1-ETO fusion gene, which is generally associated with a favorable prognosis. Nevertheless, in line with recent reports based on mutation studies of the c-kit, FLT3, and Ras genes, we have also experienced poor clinical outcomes in some patients with CBFA-AML, with rapid relapse of patients within a few months of conventional intensive chemotherapy with cytosine arabinoside or even autologous stem cell transplantation (SCT) for patients in complete remission (CR). A few studies have used SCT as part of the therapeutic approach for CBFA-AML. To study the role of SCT, in light of the advances of transplant techniques, we performed a prospective study combining SCT with a novel reduced intensity conditioning (RIC) regimen. There were 10 men and 3 women with a median age of 42 years (range 17–50). Seven patients were categorized as CBFA-AML with additional cytogenetic abnormalities. Two patients had either FLT3-ITD or c-kit mutations. All CBFA-AML patients in CR received one cycle of consolidation chemotherapy before undergoing allogeneic SCT. Four patients had HLA-matched unrelated donors and the other donors were HLA-matched siblings. The preparative regimen comprised fludarabine 30 mg/m2 on days–6 to –2, intravenous busulfan (Busulfex, Kirin-Brewery, Japan) 3.2 mg/kg/d on days–5 to –4, and fractionated total body irradiation (TBI) 200 cGy × 2 on day–1. Cyclosporine A (CsA) and a short course of standard dose methotrexate were used to prevent graft-versus-host disease (GvHD). If no acute GvHD occurred, the CsA was tapered over 4~8 weeks with the intent to discontinue by 3 to 5 months. All patients were engrafted successfully. Eleven patients are alive at a median follow-up of 12 months (range 3–18). Four (31%) out of 13 patients developed acute GvHD, and none were grade >II. Six patients (46%) developed chronic GvHD and three of them were extensive type. Of the three patients who died, two received SCT from HLA-matched sibling donors and died due to relapse at 8 and 10 months post-transplant, respectively; the other, who had received SCT from an HLA-matched unrelated donor, died at 7 months due to sepsis combined with extensive chronic GvHD in CR. The estimated 2-year overall survival and event-free survival rates were both 77%. We suggest that allogeneic SCT with this novel RIC for heterogeneous CBFA-AML patients is a feasible option with no detrimental post-transplant complications and less frequent relapse. Further, well-designed studies with more patients, comparing our regime to conventional high-dose chemotherapy, are needed to confirm these findings.
T-Cell Depleted Unrelated Donor Stem Cell Transplants Appear to Be of Value for Adult Philadelphia Chromosome Negative ALL Patients and Should Be Evaluated Prospectively in New Large Group Studies
Authors: Patel B. R , Kirkland Keiren , Pearce R. , Clark Richard E , Craddock Charles F. , Liakopolou Effie , Fielding Adele K , Mackinnon Stephen , Olavarria Eduardo , Potter Michael N , Russell Nigel H. , Shaw Bronwen E. , Goldstone Anthony H. , Marks David I
Published: April 2020

Matched sibling allogeneic haematopoietic stem cell transplantations (HSCT) in first complete remission (CR1) result in superior survival compared to standard chemotherapy in adult patients with acute lymphoblastic leukaemia (ALL). However, application is limited by donor availability. The role of unrelated donor (URD) transplantation in ALL in CR1 is not clearly delineated. We report a retrospective series of URD transplants in 55 adults with Philadelphia chromosome (Ph) negative acute lymphoblastic leukaemia (ALL) in CR1 reported to the British Society of Blood and Marrow Transplantation registry from 1993 to 2005. Median follow-up of survivors was 55.2 months (range 17–160). Median age was 25.5 years (range 15.7–50). Eighty nine percent of patients had at least one adverse prognostic feature. Sixty six percent of transplants were matched at 10 of 10 loci and 34% were mis-matched. T cell depletion (TCD) was carried out by in-vivo Campath administration in 96% of recipients with additional ex-vivo TCD in 21% of these patients. The estimated actuarial Overall survival (OS), Disease free survival (DFS) and non relapse mortality (NRM) were 59%, 57% and 19% at 5 years respectively. The overall incidence of Grade II–IV and III–IV GVHD was 25% and 7% respectively. The actuarial estimate of extensive chronic GVHD was 22 % at 5 years. HLA mismatching at 2 alleles or antigens was associated with poorer survival (RR=3.22, (1.08–9.64). T depleted URD HSCT can result in good OS and low TRM for adults with high risk ALL in CR1 and merits prospective comparison with other methods of GVHD prophylaxis.
Busulfan and Cyclophosphamide for Hemopoietic Stem Cell Transplantation from Related and Unrelated Donors in Patients with Myelodysplastic Syndrome
Authors: Shi Jimin , Huang He , Cai Zhen , Luo Yi , Zheng Weiyan , He Jingsong , Xie Wanzhuo , Li Li , Ye Xiujin , Zhang Jie , Lin Maofang
Published: April 2020

The myelodysplastic syndromes (MDS) are clonal hemopoietic disorders, characterized by ineffective hemopoiesis resulting in single or multilineage peripheral blood cytopenias, dysplastic morphology in single or multiple lineages, and, in many patients, clonal cytogenetic abnormalities. Hematopoietic stem cell transplantation is currently the only therapeutic modality that is potentially curative. A total of 17 patients (aged 16–40 years; median, 26 years) with MDS were treated with busulfan (BU 3.2 mg/kg/d, −7 d `−4 d,) plus cyclophosphamide (CY, 60 mg/kg, −3 d `−2 d)and hemopoietic stem cell (HSC) transplantation from related (n =5) or unrelated donors (n =12). At the time of transplantation, 13 patients had less than 5% myeloblasts in the marrow, and 4 patients had more advanced disease. Mycophenolate mofetil combined with cyclosporin A and methotrexate was used for prevention of acute graft-versus-host disease after transplantation. Lipo prostaglandin E1 was used in prophylactic regimen for hepatic venoocclusive disease. All but 2 evaluable patients had engraftment. Neutrophil count began to be higher than 0.5×109/L on 15 days after BMT. Platelet count began to be higher than 20×109/L on 18 days after BMT. The Kaplan-Meier estimates of 3-year relapse-free survival (RFS) were 57%, and only one patient relapsed after six months of transplantation. Non relapse mortality (NRM) at 100 days was 12%. Thus, a targeted BUCY regimen provided effective transplant conditioning for patients with MDS receiving transplants from HLA identical siblings or alternative donors.
Timing of G-CSF Injection for Effective Autologous Stem Cell Collection
Authors: Kim Jeong-Eun , Suh Cheolwon , Choi Yunsuk , Kim Eun Kyoung , Sohn Byeong Seok , Park Inkeun , Yoon Dok Hyun , Yoo Changhoon , Jang Geundoo , Kim Shin , Lee Dae Ho , Kim Sang -We , Lee Jung Shin
Published: April 2020

Granulocyte colony-stimulating factor (G-CSF) has been given approximately 12 hours before leukapheresis in the patients who are planned to do autologous stem cell collection. The objective of this study was to identify better timing of G-CSF administration which improves the efficacy of autologous stem cell collection. A total of 262 patients (157 male and 105 female patients, ages from 15 to 66 years with median of 49 years) who underwent autologous stem cell collection from January 2000 to March 2008 were included. The patients were diagnosed with Hodgkin’s lymphoma (N=19), non-Hodgkin’s lymphoma (N=131) and multiple myeloma (N=112). Peripheral blood stem cells were mobilized with lenograstim (Choongwae Pharma Corp., Seoul, Korea) following chemotherapy, such as cyclophosphamide (N=141), ESHAP (Etoposide, methylprednisolone, Ara-C and cisplatin, N=73) with (N=14) or without rituximab (N=59) and other chemotherapy regimens (N=48). Before November 2004, patients received lenograstim injection at a dose of 10 μg/kg subcutaneously at 8:00 PM and underwent peripheral stem cell collection on next day (N=129). After then patients received lenograstim injection at 6:00 AM and underwent peripheral stem cell collection at the same day (N=133). Injection of lenograstim at 6 AM was superior to injection of lenograstim at 8 PM with greater numbers of total collected CD34+ cells/kg in shorter duration of leukapheresis procedures (13.29 × 106 versus 8.51 × 106 CD34+ cells/kg; p=0.001, 2 versus 3 leukapheresis procedures; p=0.035). The median number of CD34+ cells/kg collected at first leukapheresis was also greater in 6 AM group (3.94 × 106 versus 2.47 × 106 CD34+ cells/kg; p=0.001). Stem cell collection efficacy defined as ratio of total collected CD34+ cells per days of leukpaheresis were 5.34 and 2.96, respectively. It was significantly better in the group of patients received lenograstim injection at 6 AM (p=0.001). The median number of patients who achieved a total collected CD34+ cells > 5 × 106/kg was also greater in 6 AM group (113 versus 96; p=0.002). The present study shows that injection of lenograstim at 6 AM improves the efficacy of stem cell collection with greater number of collected CD34+ cells/kg in shorter duration of leukapheresis procedures compared to that of 8 PM group.
Immunologic Recovery after Autologous Transplant in Multiple Myeloma and Progression Free Survival
Authors: Trigo-Miranda Fernanda Maria Rodrigues , Bergantim Rui Cordeiro , Pinto Ricardo Moreira , Guimarães Patricia , Guimaraes Jose E.
Published: April 2020

Several factors influencing disease progression and survival have been identified in multiple myeloma (MM). We analysed a series of 49 consecutive patients with MM that underwent autologous haematopoietic progenitor cell transplant (HPCT) in one center regarding the following variables: use of G-CSF for haematopoietic recovery post-transplant; recovery of normal IgM levels at day +100 post-transplant; levels of lymphocytes namely of the CD4+ and CD8+ subsets also at day +100. Before 2006, all patients had G-CSF starting 24 hours after the cell infusion until neutrophil > 500×10^9/L in two consecutive days; in the years 2006–2008, no G-CSF was given to transplanted patients. A historical comparison was done and at the time of this study no significant difference in progression free survival (Kaplan-Meyer analysis), was detected between the two groups, possibly due to the shorter follow-up of the “no G-CSF” (n=19) group; nevertheless median progression free survival (PFS) in the “G-CSF” group was 12 months while median PFS was not attained in the “no G-CSF” group (median follow-up = 7 months). Post transplant IgM levels were also determined in 39 patients. Eighteen patients recovered normal IgM levels at day +100 (46.8 %) and 21 (53.8 %) did not. Comparison of Kaplan-Meyer curves for the two groups did not show any statistically significant difference but there is a sharp difference between median PFS of the “low IgM” (10 months) and the “normal IgM” (27 months) groups. CD4/CD8 ratio was determined in 18 patients at day +100. The ratio varied between 0 and 0.63 (median – 0.305). No correlation was found between post-transplant IgM recovery and CD4/CD8 ratio. In conclusion, in our series of MM patients treated with autologous HPCT we could not find a definite relationship between immunologic recovery and response to treatment although there is a trend to a better outlook of the patients which recover normal IgM levels. It is also uncertain whether use of G-CSF in the post-transplant period would have any effect on disease behaviour.
Significance of Rising Levels of Minimal Residual Disease in Patients with Philadelphia Chromosome-Positive Chronic Myelogenous Leukemia (Ph+ CML) in Complete Cytogenetic Response (CGCR)
Authors: Kantarjian Hagop M , Shan Jianqin , Jones Dan , O’Brien Susan , Rios Mary Beth , Jabbour Elias , Cortes Jorge
Published: April 2020

Background . Patients with Ph+ CML receiving tyrosine kinase inhibitors (TKIs) are frequently monitored for response by quantitative polymerase chain reaction (QPCR) studies for minimal molecular disease. The clinical significance of rising levels of QPCR in CGCR is uncertain.

Study Aims . To evaluate the relevance of increases of QPCR levels in patients with CML in CGCR on therapy.

Study Group and Methods . Of 258 patients on imatinib therapy for newly diagnosed CML, 116 patients in durable CGCR on imatinib therapy for at least 18 months had significant QPCR increases (documented at least twice) as defined by literature reports. These were analyzed by the achievement of major molecular response (MMR; QPCR < 0.05%), and by the degree of QPCR increase.

Results. The outcome of patients by disease status (still in MMR vs. loss of MMR vs. never in MMR) and by the QPCR level increase are shown in the Table. Only 13 of 116 patients (11%) with significant QPCR increases had CML progression; 11 of them were among 44 patients (25%) who either lost a MMR or never had a MMR, and had > 1 log increase of QPCR. The 5-year survival of all 116 patients was 92%, suggesting the minimal relevance of QPCR increases in patients in CGCR.

Conclusion . Most patients with significant QPCR increases remain in CGCR. Patients who lose a MMR or never achieve a MMR, and have > 1 log increase of QPCR, should be monitored more closely, and may be evaluated for mutations of BCR-ABL kinase domain and considered for investigational therapeutic interventions. Allogeneic stem cell transplant should not be considered in view of the excellent survival.

Outcome of Patients in CGCR by QPCR Increases

Disease Status QPCR Log increase No. Patients CML Progression Median follow-up from QPCR increase in months (range) Persistent MMR Any 28 0 36 (3–62) Loss of MMR >0.5–1 12 0 34 (14–59) >1–2 25 3 31 (6–52) >2 11 4 45 (20–57) Not in MMR <1 32 2 35 (10–70) >1 8 4 25 (12–56)
The Alternative Breakpoint Region (ABR) of BCL6 Is An Active Transcribed Region in Germinal Center B Cells and May Serve as An Enhancer
Authors: Shen Yulei , Ramachandrareddy Himabindu , Chan Wing C. , McKeithan Timothy
Published: April 2020

Chromosomal translocations at 3q27 involving BCL6 occur in two different regions. Rearrangements with breaks within the major breakpoint region (MBR), comprising the first exon and part of the first intron of BCL6, are among the most common genetic abnormalities in of B-cell non-Hodgkin lymphoma, whereas breaks within an alternative breakpoint region (ABR), located between 245 and 285 kb 5′ to BCL6, have also been reported in follicular lymphoma grade and a small group (6.4%) of diffuse large B-cell lymphomas (DLBCLs). As a result of the MBR translocation, BCL6 expression is deregulated by promoter substitution with either immunoglobulin (Ig) genes or non-Ig genes as partners. A role for deregulated BCL6 expression in the pathogenesis of DLBCL has previously been confirmed in a mouse model. However, the biological role of the more distant ABR region is still not known. Using real-time PCR, we identified in the ABR region an evolutionarily conserved DNase I hypersensitive site (named Far5) which contains a conserved composite binding site for transcription factors PU.1 and IRF4, both of which play important roles in B-cell differentation. Further studies demonstrated that chromatin in the Far5 region, 190kb upstream of BCL6 promoter, has an open configuration in DHL6, Granta 519 and U266 cell lines. Far5 DNA showed enhancer activity by a luciferase reporter assay. PU.1 binds to Far5 in vivo (DHL16 cell line) by a chromatin immunoprecipitation (ChIP) assay, and PU.1 binds in vitro to the conserved PU.1/IRF composite site in Far5 synergistically with either IRF4 or IRF8. ChIP-on-chip assays showed Far5 histone H3K4 monomethylation, a chromatin modification associated with gene enhancers and other regulatory elements. In addition, we identified a transcript upstream of the Far5 region that is specifically expressed in germinal center (GC) B cells, but not at other stages of B-cell differentation. These results indicated that Far5 may play a role in selective expression of intergenic transcripts in GC B-cells. In other genes, intergenic transcription plays a role in looping between distal regulatory regions and the promoter. Our data showed that ABR region is constitutively active in GC B-cells and may play an important role in increasing BCL6 transcription when naïve B cells differentiate into GC B-cells. Further investigation of interactions between the ABR and the BCL6 promoter will uncover the regulatory function of the BCL6 ABR in B-cell differentiation and B-cell lymphomas.
Expression of Circadian Core Clock Gene Per2 Is Markedly Down-Regulated in Diffuse Large B-Cell Lymphoma
Authors: Thoennissen Nils Heinrich , Cao Qi , Doan BaoNgan , Abbassi Sam , Nowak Daniel , Müller-Tidow C. , Said Jonathan , Gary Sigal , Koeffler H. Phillip
Published: April 2020

Non-Hodgkin’s lymphoma (NHL) is a heterogeneous clinicopathologic entity characterized by distinct cells of origin, cytogenetic and molecular aberrations. In vivo studies recently showed that mice deficient in Period (PER) 2 developed lymphomas. Furthermore, we and others have recently suggested that the core circadian clock genes Period (Per)1 and Per2 have been linked to DNA damage response pathways. We showed that both genes are involved in tumor suppression by regulating cell cycle- and apoptosis-related genes. In mammals and other vertebrates, heterodimers of the basic helix-loop-helix-PAS transcription factors, CLOCK and BMAL1, activate transcription of the Period and Cryptochrome (Cry1 and Cry2) genes via binding to E-box sequences in their promoters. Subsequently, the PER and CRY repressor proteins accumulate in the nucleus and inhibit CLOCK:BMAL1 complexes, thereby inhibiting their own gene expression. This forms the major negative circadian feedback loop. In the present study, we focused on Per1 and Per2 and their role in different types of NHL. Real-time reverse transcriptase-polymerase chain reaction (real time RT-PCR) was performed to determine the mRNA expression levels of Per1 and Per2 in patients with diffuse large B-cell lymphoma (DLBCL; n = 37), mantle cell lymphoma (MCL; n = 12), follicular lymphoma (FL; n = 12), and Burkitt’s Lymphoma (n = 6) compared to human normal tonsil samples (n = 10). We further tested their expression in 14 different human lymphoma cell lines. The relative expression of Per2 was markedly down-regulated in all DLBCL samples compared to normal tonsils (mean fold change: − 20.5 ± 28.7; p < 0.001). On the other hand, levels of expression of Per2 in samples of either MCL (2.5 ± 2.6; p = 0.1), FL (1.1 ± 10; p = 0.9), or Burkitt’s Lymphoma (− 1.1 ± 2; p = 0.07) showed no significant change compared to the normal tonsils. Moreover, we found no significant regulation of the mRNA levels of Per1 in any of the four NHL subtypes. We confirmed our results by showing concordant results in human lymphoma cell lines. Again, Per2 expression levels were persistently down-regulated in the 7 DLBCL cell lines (OCI-Ly1, -Ly4, -Ly7, -LY10, and SUDHL-4, -6, -16; mean fold change: − 8.6 ± 5.7; p < 0.001) compared to normal tonsils, but not in MCL (SP-49, Jeko-1, NCEB-1), FL (FLK-1), Burkitt’s lymphoma (Daudi), and B-precursor acute lymphoblastic leukemia (Nalm-6, BALL-1) cell lines (p > 0.05). Western blot protein analyses showed easily detectable levels of PER2 in tonsil samples, but the protein was either not expressed or significantly reduced in expression in human DLBCL cell lines. In summary, our results strongly suggest for the first time that the disruption of the key circadian clock gene Per2 may play a role in the development of DLBCL.
The Research of Phenylhexyl Isothiocyanate’s Influence on p16 Gene Methylation of Leukaemia Cell
Authors: Chen Bao-An , Shou Bei-ming , Zhou Dong-rui , Liu Delong , Ding Jia-hua , Sun Yun-Yu , Wang Jun , Cheng Jian , Zhao Gang , Song Hui-hui , Bao Wen , Wang Jue-qiong , Yuan Peng , Pei Xiao-ping , Chen Ning-na , Ma Xu-dong , Lu Zu-hong , Lu Quan-yi , Lai Yong-rong , Huang He
Published: April 2020

Objective Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. The aim of this study was to study whether phenylhexyl isothiocyanate can reduce the methylation level of P16 gene. This study used a microarray-based method for quantificationally detecting changes of P16 gene methylation in leukemia patient and U266 cell line. And to simply discuss the effect of phenylhexyl isothiocyanate on tumor methylation.

Methods This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. One set of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of P16 gene CpG islands. Each set contained a pair of methylated and unmethylated oligonucleotides for interrogating 3 CpG sites in close proximity. By TA cloning, PCR, sequencing, positive and negative DNA targets were required. Next drawing a standard curve by fluorescence intensity. Leukemia samples DNA were abstracted and bisulfite-modified. Sample DNA targets were required by PCR amplification and were hybridized with the microarry. Finally the microarry was scanned with ScanArray Lite microarray analysis systems.

Results The linear relationship (R2=0.9660) was established and it could be used to eliminate background noise. The methylation level of U266 is hypermethylation, after cultured with PHI, the level reduce. Eleven patients have P16 gene hypermethylation in thrity. After culture with PHI, seven patients showed level reduce, one patient showed raise, three patients showed remaining.

Conclusion PHI can reduce the methylation level of P16 gene in U266 cell line, and also can reduce the methylation level of P16 gene of leukemia patient samples in vitro culture.
Rare Congenital Bleeding Disorders from a Tertiary Haematology Referral Center in Western India
Authors: Kelkar Anjali Jaydeep , Apte Shashikant , Melinkeri Varsha , Melinkeri Samir , Karpe Ashay , Bhave Saurabh , Sayaji Bhavna
Published: April 2020

Introduction: Bleeding disorders are present in varying frequency in a given population. We present our data of rare bleeding disorders (excluding Haemophilia & von Willebrand’d disease) since little data about the same is available from developing countries. Many of the rare bleeding disorders are associated with a strong history of consanguinity, the frequency of consanguineous marriages are between 12 –42 %.

Materials and Methods: These cases were part of the referral for evaluation of congenital bleeding disorders; some of the cases were diagnosed as part of pre-operative coagulation screen. All the cases were subject to a detailed history encompassing the timing site and frequency of bleed, history of consanguinity, umbilical cord bleed. Lab. evaluation included bleeding time (Ivy’s method), clotting time., in vivo adhesion and clot retraction. Prothrombin and activated partial thromboplastin time with correction studies as and when required, Factor XIII screen with urea solubility test. Complete blood count was done prior to bleeding time and platelet adequacy was confirmed. All blood samples used for coagulation assays were collected in sterile citrate vaccutainers. Further investigations were performed including factor assays depending upon the results of the basic profile.

Results: Feb 2000 till August 2008.

Total No. of cases studies I from Year 2000 970 Abnormal cases 75 Consanguinity 25 Disorder No. Of Cases Factor V Deficiency 5 Factor VII Deficiency 4 Factor X Deficiency 4 Factor XI Deficiency 1 Factor XII Deficiency 6 Factor XIII Deficiency 8 Combined factor deficiency 4 Factor I deficiency 11 Dysfibrinogenemia 6 Factor II deficiency 1 Glanzmann’s thrombasthenemia 25 Bernard soullier’s syndrome 2

Conclusion: In our institution which caters to a large population in and around city of Pune, Maharashtra Western India, we have detected a good number of patients with rare bleeding disorders. These disorders can be found on a more frequent basis as we have a significant incidence of consanguineous marriages, as our data shows i.e approx. 33%. This report highlights need for extended family search and molecular characterization so as to make antenatal diagnosis amenable for severe bleeding disorders.
Use of Factor VIII/Von Willebrand Factor Complex for Inducing Immune Tolerance in Hemophilia a Patients Who Have Failed with Recombinant Fviii: Recent Canadian Experience
Authors: Vincent Anne-Marie , Chan Anthony , Israels Sara J. , Poon Man-Chiu , Wu John K. , Rivard Georges E.
Published: April 2020

Immune tolerance induction (ITI) is effective in approximately 63–83% of hemophilia patients with inhibitors. Poor prognostic factors are age > 18 years, ITI started > 4 years after inhibitor development, inhibitor peaks > 200 BU, inhibitor titre > 10 BU at ITI initiation, and previously failed ITI. It has been reported that patients who have failed ITI with a recombinant FVIII concentrate achieved and maintained immune tolerance when VWF/FVIII complex concentrates were used. We present our Canadian experience with a factor VIII/von Willebrand factor concentrate (Humate-P) for inducing immune tolerance in patients who have previously failed ITI. A questionnaire was sent to all Hemophilia Treatment Centers of Canada. Data collected included baseline FVIII level, mutation, date at initial diagnosis of inhibitor, peak titer, concentrate associated with development of inhibitor, inhibitor titer at initiation of first ITI, concentrate and regimen used, and concentrate, dosing and outcome of secondary ITI with Humate-P. All Hemophilia Centers agreed to participate. 5 Centers submitted data about 8 patients. One patient resistant to initial ITI with Kogenate FS achieved good partial response with Humate-P, with titers going down from 40 to values between 0,6 and 4 BU. One patient who responded to initial ITI with Kogenate FS with peaking of inhibitor to 380 BU was remaining above 100 BU and lowered to 28 BU on Humate-P. In three patients, Humate-P induced an anamnestic response. Two of those patients had a concomitant line infection at time of switching to Humate-P. The third of those three patients had a transient anamnestic response after introduction of Humate-P. One patient had successfull induction of immune tolerance using multiple therapies (cyclophosphamide, Immunate, IVIG, Rituximab) and maintained remission under Humate-P. One patient was on ITI for 27 months using Recombinate and switched to Humate-P when Recombinate was withdrawn from the market. After 12 months, the inhibitor had gone from 12 to 7.3 BU. One patient attained complete remission during primary ITI using recombinant FVIII concentrate (Advate) and maintained remission when switched to Humate-P. In subjects failing ITI with recombinant factor VIII concentrates, Humate-P may be considered as a second line for inducing immune tolerance.
Incidence of Suicide in U.S. Hemophilia Patients
Authors: Gomperts Edward D. , Presley Rodney J , Baker Judith , Holtz Jean
Published: April 2020

The causes of death in hemophilia have changed in the USA over the past few decades. Prior to the 1970’s, individuals with hemophilia were succumbing to the seriousness of the hemorrhagic disorder with bleeds being an important cause of mortality. However, with the advent of clotting factor concentrates and the introduction of comprehensive care, a hemorrhagic cause of death became less likely with life expectancy rising to almost that of the non-hemophilic population in the early 1980’s. The onset of the HIV epidemic changed this encouraging statistic, with more than 60% of HIV-infected individuals with hemophilia succumbing to this virus to date. Hepatitis B and C have also taken their toll. However, prior to the HIV epidemic, during the peak period of the epidemic and in the post-HIV period, health care providers continue to express concern about deaths due to suicide. Since 1998 the 135 federally funded Hemophilia Treatment Centers (HTC) have enrolled over 13,000 males with hemophilia, representing over 80% of hemophiliacs who receive care at these HTCs, in the Universal Data Collection (UDC) surveillance program. We used data on all deaths among UDC enrollees, as well as non-UDC enrollees at the HTCs to estimate the incidence of suicide over the nine year period of surveillance. Data from completed mortality forms of both UDC and non-UDC enrollees were analyzed to determine the incidence of suicide episodes for the period 1998 through 2007. There were a total of 17 suicide deaths over this time, producing a crude rate of 12.5/100,000 person years, compared with the rate for the US male population of 17.7/100,000. Because the hemophilia population is younger than the general population, age adjusted suicide rates were also calculated. Detailed age distribution data were available only for persons enrolled in UDC. Therefore, adjusted rates were based on 8 suicides among the 554 deaths that were reported for persons enrolled in UDC. All 8 suicides occurred among White non Hispanic males between the ages of 20 and 61 (Mean =36.1 years). Three of the persons who died from suicide were HIV positive, one was HIV and HCV positive, one HCV and HBV positive, and one was HCV positive. The crude rate of suicide for persons enrolled in UDC was 16.1/100,000 persons years. Using the indirect age adjustment method the adjusted rate is 11.5/100,000 person years, based on 2004 census data, resulting in 5.8 predicted deaths. The observed and expected numbers of deaths were not statistically significantly different. The available data does not indicate a suicide mortality rate higher among hemophilia patients receiving care at HTCs enrolled in UDC than the US male population.
PK Comparability of VWF/FVIII Concentrates after Repeated Dosing
Authors: Kronthaler Ulrich , Stockschlaeder Marcus , Dickneite Gerhard
Published: April 2020

Introduction: Appropriate correction of hemostasis in VWD is essential not only to stop acute perioperative bleeding, but also to promote postoperative wound healing. In order to prevent a drop of the VWF plasma level below the hemostatically necessary concentration, repeated infusions of VWF containing concentrates are required. Plasma derived VWF/FVIII concentrates vary, however, in several aspects such as the VWF/FVIII ratio. As therapy of VWD primarily aims at correcting functional VWF level, the question arises, whether VWF/FVIII concentrates differ with regard to the plasma level of FVIII resulting from repeated concentrate infusions.

Methods: The goal of the present study was to compare VWF and FVIII levels after repeated administration of 150 IU (VWF:RCo)/kg (7 doses at 4-hour intervals). Two plasma derived VWF/FVIII concentrates, Humate-P®/Haemate® P and Wilate®, were selected for the comparison. The concentrates were administered as i.v. bolus to CHB rabbits (n=6/group). Blood samples were collected for close monitoring of the resulting substitution levels. The VWF and FVIII levels were quantified using commercially available ELISA assays.

Results: Pharmacokinetic analysis (AUC, Cmax, half-life, MRT, clearance) demonstrated that the time course of VWF plasma levels were similar. The ratio of the VWF AUC of Humate-P®(Haemate® P)/Wilate® was 0.98 and the maximal VWF plasma concentration of Humate-P®/Haemate® P was slightly higher, at a ratio of 1,16. In contrast, the FVIII levels continuously accumulated upon treatment with Wilate® leading to trough levels, which were about 1,6 fold higher compared to Humate-P®/Haemate® P (Figure). Accordingly, also the peak FVIII level was 1,7 fold higher upon treatment with Wilate®, while the half-life of FVIII did not differ substantially between the two concentrates. The FVIII trough level of Wilate® together with its higher peak level resulted in an AUC, which was 2 fold higher (p<0.0001) than observed for Humate-P®/Haemate® P.

Conclusion: Against the background of Wilate® containing a two fold higher FVIII concentration compared to Humate-P®/Haemate® P, such differential effects appear plausible. At nominally equal VWF:RCof repeated doses, the associated AUC with respect to FVIII:Ag of Humate-P®/Haemate® P was significantly lower, as compared to Wilate®. This is obviously due to the lower FVIII content in the Humate-P®/Haemate® P product. Therefore, a lower accumulation of FVIII trough levels was observed for Humate-P®/Haemate® P as compared to Wilate®.

Figure Figure
Interaction Between Oral Contraceptive Use and Coagulation Factor Levels in Deep Vein Thrombosis
Authors: Zotz Rainer B. , Kloeckner Simone , Scharf Rudiger E. , Gerhardt Andrea
Published: April 2020

Background: Since not all women using oral contraceptives (OC) develop deep vein thrombosis (DVT), the presence of additional risk factors in women with DVT and OC use is likely. High levels of coagulation factor activities are established risk factors of DVT. There are, however, limited data on the combined effect of OC use and high levels of procoagulant coagulation factors, a shortened aPTT or markers of inflammation like erythrocyte sedimentation rate (ESR) or CRP.

Materials and Methods: 246 women with a first DVT (123 associated with OC use) and 246 normal women matched for age and date of birth were evaluated. Cut-off values for continuous variables were the 90% percentile determined in normal women. OC use influences the levels of many coagulation factors. Using the mean effect of OC use on coagulation factor levels (measured in the control subjects), we estimated the coagulation factor levels in patients prior to the index date, i.e. the time of the thrombosis.

Results: Use of OC was associated with an increase in risk of 3.0. This risk increased due to interaction with factor I (FI) (Odds Ratio (OR) 14.2, p=0.0001), FVIII (OR 5.1, p=0.0001), FIX (OR 5.5, p=0.0001), FXI (OR 12.0, p=0.0001), vWF:Ag (OR 7.7, p=0.0001) and aPTT (OR 20.2, p=0.004). In contrast, no significant increase in risk was found when OC use was associated with high levels of ESR or CRP.

Conclusion: The results of this study indicate that the joint effects of OC use and high coagulation factor levels increase the risk of DVT. This risk increase appears to be of clinical relevance particularly in individuals with shortened aPTT values or high levels of fibrinogen.
First Census of Patients with Hemophilia a/B and Von Willebrand Disease Type 3 in the Eastern Part of Germany since 1988
Authors: Koscielny Juergen , Scholz Ute , Klamroth Robert
Published: April 2020

Diagnostics and treatment of severe forms of haemorrhagic diatheses (HD), such as haemophilia A, B or type 3 von Willebrand disease (VWD) need high standards in haemophilia treatment centers (HTCs). Due to their generally low incidence, a close cooperation of haemophilia treaters is needed to optimize treatment strategies. For this purpose, the “Kompetenznetz Hämorrhagische Diathesen Ost” was founded. The first project was the conduction of a survey of epidemiological data of patients with HA, HB and type 3 VWD in all HTCs in the eastern part of Germany. The study assessed the data regarding numbers of patients treated with HA, HB or type 3 VWD, disease severity, regime of coagulation factor replacement (CFR) and the frequency of factor VIII and IX inhibitors. Data of 838 patients (a prevalence of 52.38 per 1 million citizen) from 27 HTCs were evaluated (5–151 per HTCs). Among the included patients, 201 (prevalence: 12.56/million) were children or adolescents (< 18 years). 81 children suffered from severe HA (prevalence: 5.06/million), 20 from severe HB (prevalence: 1.25/million) and 10 from type 3 VWD (prevalence: 0.63/million). In 637 adults (prevalence: 39.82/million) we found 246 with severe HA (prevalence: 15.38/million), 51 severe HB (prevalence: 3.19/million) and 28 VWD of type 3 (prevalence: 1.75/million). The most commonly used treatment of CFR in patients with severe disease was prophylactic modality. 90% of the children received prophylaxis, but also in 64% of the adults an intermittent or longterm secondary prophylaxis was the preferred treatment. At the time of survey conduction, a factor VIII or IX inhibitor was present in 1.9% of the children and in 1.6% of the adults with haemophilia. Our data allow an overview of the number of patients and treatment strategies in the eastern part of Germany.
Complement Deposition Is Predictive of a Low Absolute Immature Platelet Fraction in Immune Thrombocytopenic Purpura
Authors: Andemariam Biree , Bussel James B , Peerschke Ellinor I.B.
Published: April 2020

The role of platelet-associated complement in the pathophysiology of immune thrombocytopenic purpura (ITP) is not well understood. Although various humoral and cell mediated mechanisms have accounted for the decrease in platelet count, the role of the complement system has not been well defined. To test this, we examined complement fixation on platelets using plasma of 100 ITP patients and 50 healthy volunteers. In situ complement fixation was measured as C1q deposition on heterologous platelets using an ELISA approach. Deposition of C1q was evaluated using specific primary monoclonal antibodies (Quidel, San Diego, CA), and an alkaline phosphatase conjugated goat anti mouse secondary antibody and p-NPP substrate to quantify primary antibody binding. Substrate conversion was measured at 405 nm and was proportional to antigen deposition (complement component) on the platelet surface. Controls for specificity included wells coated with blocking solution only, and platelets exposed to buffer instead of plasma. Results for patient samples were normalized relative to an internal assay control (pooled normal plasma) and expressed as a ratio of C1q deposition. Ratios equal to or greater than 1.9 were considered positive for complement fixation. No significant relationship was observed for complement fixation when samples were stratified by gender, splenectomy status, prior treatment or total platelet count. However, a significant relationship between Absolute Immature Platelet Fraction (A-IPF) (Sysmex, Kobe, Japan) and C1q deposition was observed. The data suggest that a positive complement fixation test defined by C1q deposition is predictive of a low A-IPF (<15) with a positive predictive value and specificity of 100%. The two tailed Fisher exact test gives a p-value of 0.0265. In conclusion, these data suggest that complement fixation in the classical pathway may play a role in reducing the number of immature platelets in patients with ITP, perhaps by a direct effect of complement deposition on megakaryocytes and therefore anti-complement therapy may have clinical utility. Further studies that include an examination of complement fixation in the bone marrow, especially on megakaryocytes, may elucidate the exact mechanism of this destruction. Alernatively, flow cytomety of immature platelets to compare them to mature platelets in regard to complement deposition would be of interest.
Elevated Expression of IL-17 and IL-23 in Patients with Immune Thrombocytopenic Purpura
Authors: Li Feng , Zou Shanhua , Cheng Yunfeng
Published: April 2020

Introduction: In conjunction with IL-6 and TGF-β, IL-23 stimulates naïve CD4+ T cells to differentiate into Th17 cells. Th17 cells produce IL-17, a pro-inflammatory cytokine that play an important role in the pathogenesis of several autoimmune disorders. However, to date, the role of Th17 cells in immune thrombocytopenic purpura (ITP), a type of autoimmune diseases, has not been clearly established yet.

Methods: Peripheral bloods were obtained from 10 patients with ITP at onset, in remission and from 15 healthy control subjects. The frequencies of IL-17 producing T cells in peripheral blood were analyzed by flow cytometry. Peripheral blood Mononuclear cells (PBMCs) were isolated using Ficoll density-gradient centrifugation and the CD4+ cells were separated by immuno-magnetic microbeads selection. Plasma concentrations of Th17 cell-associated cytokines such as IL-12, IL-17, IL-23, IFN-γ, IL-6 and TGF-β were measured using ELISA. The mRNA expression levels of IL-17, IL-12p40, IFN-γ, IL-23p19 in CD4+ cells were determined by Real-Time PCR.

Results: The frequencies of IL-17-producing T cells were significantly increased in ITP patients at onset, compared to ITP patients in remission (10.7±5.5 % vs 4.1±3.5 %, p < 0.05) and healthy controls (10.7±5.5 % vs 2.1±1.6 %, p < 0.05), however there was no statistical difference between ITP patients in remission and healthy controls. Comparing to healthy subjects, the plasma concentrations of IL-12 (33.3±15.25 pg/ ml vs 12.8±7.24 pg/ml, p<0.05), IL-17 (37.3±12.1 pg/ml vs 5.1±3.6 pg/ml, p < 0.05), IL-23 (30.01±9.33 pg/ml vs 10.42±13.19 pg/ml, p < 0.05) in patients with ITP at onset were found significantly elevated whereas no statistical difference was observed for the levels of IL-12, IL-17 and IL-23 between ITP patients in remission and healthy controls. Furthermore, the expression levels of IL-23p19 mRNA were significantly increased in ITP patients at onset, compared to healthy controls. Changes in IL-23p19 mRNA expression and IL-17 were strongly correlated (R = 0.66, p < 0.05).

Conclusion: Our results support the hypothesis that Th17 cells are involved in the development of ITP and Th17 cells could potentially constitute a novel therapeutic target.