bluuebunny/crossref_metadata_2025 huggingface dataset:
Common Submicroscopic Genomic Imbalances Accompany the Ph Chromosome at Diagnosis in Chronic Myeloid Leukemia
Authors: Khorashad Jamshid S , De Melo Valeria A , Fiegler Heike , Gerrard Gareth , Brudzevsky Dan , Marin David , Apperley Jane F. , Goldman John M , Foroni Letizia , Reid Alistair G
Published: April 2020

Despite the presence of a consistent genetic abnormality, CML patients display considerable clinical heterogeneity, the basis of which is poorly understood. We therefore used a novel ultra-high-resolution genomic screening assay to search for additional acquired genomic abnormalities that might explain this clinical heterogeneity and help to assess prognosis for individual patients. Comparative genomic hybridisation (CGH) was performed with a 2.1 million oligonucleotide array on DNA extracted from presentation samples of 20 previously untreated CML patients. Each DNA sample from diagnosis was competitively hybridized against the same patient’s remission sample, which avoided detection of constitutional polymorphic copy number variants (CNVs). For representative copy number aberrations (CNAs) the CGH result was confirmed by fluorescence in situ hybridization or quantitative real-time PCR. All 20 CML patient samples harbored detectable genomic imbalances with a median of 16 CNAs per patient (range: 4–165). Of the 846 CNAs detected 679 (80%) were amplifications and 167 (20%) were deletions. 462 CNAs (55%) involved at least one known gene. The median size of CNAs was 58kb (range 9kb – 12Mb). One hundred and twenty seven different genomic regions were aberrant in 2 or more patients in the cohort. Of these recurrent CNAs, amplifications of the FOXD4L4, MAPK8IP1, DUSP1, PSTPIP1 and PBEF1 genes were among the most frequently detected; they were present in 3, 4, 5, 6 and 15 patients respectively. FOXD4L4 is a myeloid trancription factor previously implicated in leukemogenesis, and PSTPIP1 has been shown to be involved in dephosphorylation of ABL. MAPK81P1 and DUSP1 are both involved in the MAPK pathway, while PBEF1 is known to play a role in the NAD anti-apoptotic pathway. The presence of multiple genomic lesions at diagnosis supports the notion of an increased level of genomic instability in CML cells and raises the possibility that one or more aberrations in addition to BCR-ABL may dictate the CML phenotype. The considerable range in the number of CNAs identified at diagnosis may reflect a differing level of genomic instability in different patients. Furthermore the presence of same genetic imbalance in more than one patient suggests a role for specific genes in addition to BCR-ABL in the pathogenesis or progression of CML.
Circulating B-cell chronic lymphocytic leukemia cells display impaired migration to lymph nodes due to reduced LFA-1 expression
Authors: Hartmann Tanja Nicole , Grabovsky Valentin , Wang Wei , Desch Petra , Wollner Stefan , Binsky Inbal , Vallon-Eberhard Alexandra , Sapoznikow Anita , Haran Michal , Shachar Idit , Burger Meike , Honczarenko Marek , Greil Richard , Alon Ronen
Published: April 2020

Homing to secondary lymphoid organs and re-entry to bone marrow (BM) are central aspects of leukemic pathophysiology. We investigated the roles of the two major lymphocyte integrins LFA-1 and VLA-4 on B-cell chronic lymphocytic leukaemia (CLL) cells in these processes. We found that CLL cells expressed significantly reduced LFA-1 due to low beta2 integrin transcripts and displayed diminished adhesiveness to ICAM-1-expressing endothelium in vitro. VLA-4 expression was heterogenous but underwent rapid activation by the BM chemokine CXCL12. Nevertheless, CLL cells failed to transmigrate across VCAM-1, ICAM-1 and CXCL12 expressing endothelium due to their deficient LFA-1 expression. Furthermore, when injected into tail veins of immunodeficient mice, normal B cells rapidly homed to lymph nodes (LNs) in a LFA-1 dependent manner whereas CLL cells did not. CLL alike normal B lymphocytes used VLA-4 rather than LFA-1 to reenter the BM. In contrast, both normal and CLL B cells homed to mice spleen in an LFA-1- and VLA-4-independent manner. Our results suggest that CLL cells are deficient in LFA-1-dependent trafficking to LNs but residual subsets can still re-enter the BM. Integrin blocking could be therefore an efficient strategy to prevent circulating CLL cells from reaching prosurvival niches in LNs and BM and directing these cells to the spleen.
Direct Fluorescent Visualization of HSC Competitive Repopulation in Transparent Adult Zebrafish
Authors: Li Pulin , Zon Leonard I
Published: April 2020

Transplanted hematopoietic stem cells (HSCs) home to the marrow space, engraft and self-renew. The process of engraftment is difficult to study in most vertebrates because it is impossible to visualize the engraftment directly. Here we have developed competitive transplantation of fluorescent donor marrow samples into a completely transparent adult zebrafish called casper. This allows the direct visualization of engraftment and the process of HSC competition. Transplantation of zebrafish whole kidney marrow (WKM) cells, containing HSCs, progenitors and mature blood cells, can rescue lethally irradiated recipient fish and repopulate all the blood lineages. We used different GFP-/DsRed-labeled transgenic fish as donors in WKM transplantation, such as the myeloid specific Tg(mpo:GFP), or the ubiquitous expression of Tg(β-actin:GFP), and Red GloFish® to study the kinetics of engraftment. Engraftment can be visualized with a simple fluorescent microscope, and was evident in the recipient kidney as early as 2 weeks post transplant (wpt). By 4 wpt, mature hematopoietic cells differentiated from donor HSCs or progenitors can be observed in the recipient circulation as well as in epidermis as tissue residential macrophages. At 8 wpt, donor-derived lymphoid cells also repopulate the recipient immune system. The fluorescence intensity of engrafted cells was quantified by image analysis software and positively correlated with the engraftment efficiency quantified by FACS. Using Red GloFish® as competitor donor and Tg(β-actin:GFP) as test donor, we were able to see the competition between the two donors both by fluorescence intensity analysis and FACS analysis. To test the sensitivity of this competitive transplant system to the alteration of the ratios between the two colors, we increased the ratio of the test vs. competitor donor from 1:3 to 1:2 to 1:1. At 4wpt, in the 1:3 ratio group, only 14% of the recipients had stronger GFP intensity than the red (n=7), while the rest had weaker or equivalent GFP intensity than the red; however, this percentage increased to 43% and 50% in the 1:2 (n=7) and 1:1(n=8) groups respectively. Therefore, the relative engraftment ratio can be fast detected in vivo by direct fluorescent visualization with the transparent zebrafish recipients. Next, we treated the test donor cells with 16,16-methyl prostaglandin E2 (dmPGE2) or DMSO for 2 hours in vitro at room temperature before transplant. The engraftment efficiency of the dmPGE2 group dramatically increased by at least 2 folds compared with the DMSO group. The colors of competitor and test donors were switched, and the increase of engraftment efficiency by dmPGE2 treatment of the test donor was still evident. In conclusion, direct visualization of competitive marrow repopulation greatly facilitates the developmental, physiological or pathological study of HSCs and progenitors and should provide a new platform for the assay of specific genes or chemicals that could alter homing, engraftment or self-renewal.
A Closer Look at Cellular Iron Metabolism in IRP2 Deficient Erythroblasts.
Authors: Schranzhofer Matthias , Schifrer Manfred , Galy Bruno , Hentze Matthias W. , Mullner Ernst W. , Ponka Prem
Published: April 2020

Developing red blood cells are the major consumers of body iron which is indispensable for the enormous production of heme for hemoglobin synthesis. The uptake of iron occurs via binding of iron-loaded transferrin to its cognate receptor (TfR). Thereafter the iron is shuttled to the mitochondria where it is incorporated into protoporphyrin IX to form heme. Excess iron is enclosed within the iron storage protein ferritin. Coordinated control between iron uptake and storage is mainly achieved by the post-transcriptional regulation of TfR1 and ferritin synthesis by the iron regulatory proteins IRP1 and IRP2. Recently, two groups independently created mice lacking either IRP1 or IRP2 and showed that only IRP2 deficient mice developed microcytic hypochromic anemia. Both groups observed a reduction in TfR1 protein expression levels in the developing red blood cells of IRP2 knockout animals and suggested that the decrease in receptor levels is responsible for the development of anemia.

For a more detailed analysis of how the loss of IRP2 expression influences iron metabolism and hemoglobinization during terminal erythroid differentiation, we isolated CFU-E-like erythroid cells from mouse fetal liver of wild type, IRP1 and IRP2 knock out animals. In vitro cultivation of these primary erythroid cells and their synchronous induction for differentiation allowed us to study their cellular iron metabolism at different time points. We analyzed the extent of hemoglobinization and cell size as well as the expression of ferritin and TfR1 during various stages of erythroid differentiation in IRP1, IRP2 and wild type cells. In agreement with the published phenotype of microcytic hypochromic anemia, only erythroblasts lacking IRP2 exhibited a reduction in hemoglobinization and showed a significant increase in ferritin protein levels before and after induction of differentiation. In contrast, TfR1 protein expression levels on the cell surface were significantly decreased in IRP2 deficient cells until 24h of differentiation, but converged with those of wild type cells at 48h of differentiation at the time point at which hemoglobinization is fully in progress. Moreover, measurement of 59Fe uptake and its cellular distribution showed that there is significantly more 59Fe located in cytosolic ferritin of IRP2 knock out cells at all time points compared to their wild type counterpart. In summary, these results suggest that not only the reduced expression of TfR1, but also the up-regulation of ferritin, play important roles in the development of anemic phenotype in IRP2 knock out mice.

This work was supported by the Canadian Institutes of Health Research and the Canadian Blood Services.
T Regulatory Cells in Graft-Versus-Host Disease of the Skin
Authors: Azar Jose , Billings Steven , Schwartz Jennifer E. , Liu Yunlong , Yu Menggang , Nelson Robert P.
Published: April 2020

FoxP3+ CD25+ T regulatory (Treg) cells are known to be present in normal skin. Treg cell functional deficiency resulting in loss of suppression of activation, differentiation or expansion of effector T cells could conceivably contribute to the pathophysiology of graft-versus- host disease (GVHD). Rezvani et al. suggest that levels of peripheral blood Treg cells in donors and recipients may predict the risk of acute GVHD. Rieger et al. found a significantly lower number of Treg cells in human colonic biopsies with GVHD versus CMV colitis or normal samples. There are no reports that evaluate tissue Treg cells in human skin affected by GVHD. This study was conducted to evaluate the distribution of Treg cells in skin affected by acute GVHD versus chronic GVHD. Archived samples from patients previously reported by routine histopathological methods as acute or chronic GVHD were collected, coded and entered into a database. For immunostaining, 4-μm thick serial sections were cut and deparaffinized. Immunohistochemical stains for CD4 (Neomarkers, 1:20), CD8 (Dako, prediluted), CD25 (Dako, 1:100), and Foxp3 (Serotec, 1:250) were performed using standard techniques. The dermatopathologist evaluating the samples was blinded to the clinical outcomes. Results were scored as 0 (<10% of lymphocytes positive), 1+ (10–25% positive), 2+ (26–50% positive), and 3+ (≥50% positive). Fourteen patients with acute skin GVHD and seventeen with chronic GVHD were identified from a database of patients who had undergone nonmyeloablative allogeneic peripheral blood transplantation in the recent past. The average scores for each immunostain were calculated and are summarized in table 1. The average FoxP3 score in acute GVHD specimens was significantly lower than that in chronic GVHD specimens (average, 0.57 versus 1.41; p-value = 0.011). The average scores of CD3, CD4, CD8 and CD25 immunostains were not significantly different between acute and chronic GVHD biopsies. These findings represent the initial observation of a distinction between the distribution of regulatory T cells in acute and chronic GVHD of the skin. These observations should be confirmed in a larger sample, supported by functional assays of Treg cells, and correlated with clinical outcomes. Such studies may help to elucidate the role of Treg cells in acute and chronic skin GVHD.

Table 1.

Average score CD 3 CD 4 CD 8 CD 25 Fox P3 Acute GVHD 2.93 2.21 2.14 0.29 0.57 Chronic GVHD 2.82 2.44 2.24 0.71 1.41 p-value 1.00 0.42 0.43 0.34 0.011
Immunological Evaluation after Haematopoietic Stem Cell Transplantation for B–Thalassemia Major
Authors: Marziali Marco , Isgro Antonella , Gaziev Javid , Fraboni Daniela , Polchi Paola , Sodani Pietro , Alfieri Cecilia , Paciaroni Katia , Gallucci Cristiano , Lucarelli Guido
Published: April 2020

Background: Haematopoietic stem cell transplantation (HSCT) remains the only curative option for patients with thalassemia. After HSCT is particularly important to recover a complete immune reconstitution for prevention of opportunistic infections.

Methods: The immunological phenotype of peripheral blood mononuclear cells in 110 consecutively subjects after HLA identical HSCT performed for b thalassemia were studied. CD3+CD4+, CD3+CD8+, CD3−CD19+, CD3−CD16+CD56+ were evaluated by Flow Cytometry at 6 and 12 months after HSCT in 70 patients followed. In a part of patients CD4 naïve T cells was also evaluated at 12 months after HSCT.

Results: at 6 months after HSCT we observed reduction of the mean of CD4 T cell percentage in patients respect normal value: in mean 17, 5 % ± 9 vs 45 % ± 10 respectively. After 1 year the rate of CD4+ T-cell population progressively increase: 24 ± 9 at 12 months vs 17, 5 % ± 9 at 6 months, but the mean of CD4+ T-cell count was persistently lower than in controls. The mean of CD8 T cell is stable during the follow up and the CD4/C8 ratio decreased. The mean of the CD19 increase during the follow up (4, 5 % at 6 months vs 17% at 12 months). At 1 year after HSCT the percentage of NK (CD3−CD16+CD56+) was normalized. At 1 year after HSCT reduction of naïve CD4 T cell we observed.

Discussion. At 1 year after HSCT the immunological reconstitution is not still complete. The time and the extent of immune reconstitution depend on several factors. It is very important to continue the immune surveillance after HSTC and to evaluate other immunological parameters (i.e. thymic output) for to prevent infectious complications and elaborate an appropriate therapeutic strategy.
Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) in Stem Cell Transplant Recipients: Response to Immune Enhancement in Early Versus Late Serious Post-Transplant Infections
Authors: Safdar Amar , Georgescu Georgina , Rodriguez Gilhen , Champlin Richard
Published: April 2020

Background: The impact of GM-CSF therapy in SCT recipients with serious infection is not clear. We evaluated feasibility of GM-CSF therapy in transplant patients.

Methods: Forty-seven patients with severe infections who had received 7 or more doses of GM-CSF between 2000 and 2006 were evaluated. Response was considered at the end of GM-CSF therapy in patients with complete or partial response (CR/PR). All values are given as median ± s.d. Serious infections were defined as systemic/invasive infections requiring hospitalization.

Results: Age was 39 ± 18 years; 83% received allogeneic SCT, and 70% had leukemia. GVHD was noted in 36%; 60% of SCT recipients had cancer recurrence and 11% had graft failure. The APACHE II score was 15 ± 4. GM-CSF was given for 13 ± 12 (range, 7–57) doses. In only 1 of 5 patients with GM-CSF related adverse events (AEs), growth factor was discontinued due to persistent bone pain. Patients who received GM-CSF during late (>180 days) infections had higher CR/PR versus cytokine therapy for patients with early post-transplant infections (63% vs 35%, respectively; P = 0.06). Factors associated with treatment failure included: prior high-dose steroids (43% vs 4% in CP/PR; P = 0.01) multiple concurrent infections (65% vs 33% in CR/PR; P = 0.03), critical care unit stay (44% vs 5% in CR/PR; P = 0.001), mechanical ventilation (35% vs 4% in CR/PR; P = 0.01), serum bilirubin > 2mg/dL (30% vs 0%) in CR/PR; P = 0.004) and neutropenia (50% vs 17% in CR/PR: P = 0.02). Neutropenia was 25 ± 19 days in responders vs 34 ± 42 days in patients who failed treatment (P = 0.08). By logistic regression analysis, presence of multiple concurrent infections was associated with higher probability of death within 12 weeks of infection diagnosis (odds ratio, 4.76; 95% confidence interval, 1.1–20.5; P = 0.03).

Conclusions: Treatment with GM-CSF was tolerated without serious AEs and may improve outcomes in patients with late post transplant infections.
Prognostic Factors Associated with Outcomes of RIC and MAC Hematopoietic Stem Cell Transplantation: An Indication for the Selection of Conditioning
Authors: Kiyokawa Tetsuyuki , Nagakura Shoichi , Hidaka Michihiro , Yano Takahiro , Sunami Kazutaka , Okamura Seiichi , Uike Naokuni , Hamaguchi Motohiro , Matsui Takahiro , Horibe Keizo , Sato Kensaku , Kawakita Toshiro , Kawano Fumio
Published: April 2020

BACKGROUND: Despite recent increase of reduced intensity conditioning (RIC) transplantation, mortality rates after RIC and myeloabrative conditioning (MAC) HSCT remain high and cannot accurately predicted.

OBJECTIVE: To determine the value of pre-transplant factors in predicting one year overall survival of allogeneic HSCT with RIC and MAC. Optimizing conditioning selection based on pre-transplant prognostic factors may further improve results of allogeneic HSCT.

PATIENTS AND METHODS: A retrospective review of 415 consecutive allogeneic HSCT was performed with attention to mortality and pre-transplant factors in five hematopoietic cell transplantation centers between 2000 and 2005. Patients underwent transplantation from sibling (n=275) or unrelated donors (n=140) with MAC (n=247) or RIC (n=168). Main outcomes and prognostic factors were analyzed in multivariable analyses (a logistic regression model) with RIC and MAC.

RESULTS: Mortality before one year occurred in 252 of 415(60.1%) transplant recipients; 137 of 247(55.4%) with MAC and 115 of 168(68.5%) with RIC. Multivariate analysis identified specific and distinct prognostic factors with one year overall survival with MAC (CR/non-CR, performance status, albumin level, creatinine leve and a contain of Ara-C with preconditioning regimens) and with RIC (PS, albumin level, a contain of fuldarabin)(Table). These factors were useful for estimating survival rate of recipient after one year of transplantation (survival estimating equation).

CONCLUSION: Our results may provide indication for a better choice of conditioning regimen according to pre-transplant prognostic factors

Figure Figure Figure Figure
Prolonged Survival in Adults with Acute Lymphoblastic Leukemia (ALL) Following Reduced Intensity Conditioning and Allogeneic Transplantation
Authors: Bachanova Veronika , Weisdorf Daniel J. , Brunstein Claudio , Wagner John , Verneris Michael
Published: April 2020

Reduced intensity conditioning (RIC) and allogeneic hematopoietic cell transplantation (HCT) for adult patients with ALL may improve the tolerance and outcomes following allografting. We studied 22 consecutive patients (2001–2008) who received RIC HCT. Indications for RIC regimens included: age > 55 years (for matched related donor [MRD], >45 years for unrelated or umbilical cord [UCB]), Karnofsky index <80%, impaired organ function or prior autologous/allogeneic HCT. The median age was 49 years (24–68), median diagnostic WBC 20×109/L (1.4–248) and median comorbidity index of 3 (range: 0–8) at HCT. All patients had high risk disease, including 14 patients (64%) with Ph+ ALL, 10 patients (46%) in 2nd or subsequent remission. Five patients (23%) were treated with tyrosine kinase inhibitors pre-HCT. Uniform conditioning (Cyclophosphamide 50 mg/kg; Flu 200 mg/m2; total body irradiation (TBI) 200cGy, n=21) and graft versus host disease (GvHD) prophylaxis (Cyclosporin/MMF) was followed by MRD (n=4) or UCB donor (n=18) grafts. All patients had sustained donor neutrophil engraftment (median 10 days, range 0–28) and reached 100% donor chimerism at median day 23 post-transplantation (range 14–99 days). At 3 years, treatment related morality relapse and overall survival (OS) were 27% (95% confidence interval [CI]: 9–45%), 36% (95% CI: 14–58%) and 50% (95% CI: 27–73%), respectively. There were no relapses beyond 2 years. After a median follow-up of 33 months (range 5–77) the cumulative incidence of acute II–IV, III–IV and chronic graft versus host disease were 55%, 20% and 45%. Lower TRM for HCT in CR1 vs ≥ CR2 (8% vs. 50%; p<0.04) led to improved 3 year OS in the CR1 cohort (81% vs. 15% (p<0.01)). HCT using either MRD or UCB resulted in similar risks of GVHD, relapse and OS. These data suggest that RIC allogeneic HCT using either related or UCB donors for adults with ALL can result in modest TRM, and only limited risks of relapse and promising leukemia-free survival. Further study, especially for patients in CR1, will better define the utility of this approach for patients unsuited for more intensive, myeloablative conditioning.
Allogeneic Stem Cell Transplantation for Relapsed Hodgkin’s Disease - a Single Centre Experience
Authors: Scheid Christof , Borchmann Peter , Bredenfeld Henning , Chemnitz Jens , Holtick Udo , Cremer Birgit , Bergwelt-Baildon Michael , Hallek Michael , Engert Andreas
Published: April 2020

Hodgkin’s disease can be cured by chemotherapy in the majority of cases. However a small proportion of patients show an aggressive course with multiple relapses after chemotherapy including autologous stem cell transplantation. Whether allogeneic stem cell transplantation constitutes a valid therapeutic option for these patients remains highly controversial. We report on our experience on 9 patients with Hodgkin’s disease receiving an allogeneic stem cell transplant from matched related (n=3), matched unrelated (n=1) or mismatched unrelated (2× 9/10, 2 ×8/10, 1× 7/10) donors. Median age was 28 years (range 18 – 35). All patients were extensively pre-treated including high-dose chemotherapy with autologous stem cell transplantation in 8 of 9 cases. The disease status before transplantation was CR (n=1), PR (n=5), SD (n=2) or progressive disease in 1 case. Conditioning treatment consisted of fludarabine 30 mg/m2 day -8 to -4, melphalan 70 mg/m2 day -3 to -2 and ATG 10–20 mg/kg day -4 to -2. Ciclosporin A and short course methotrexate was used for prophylactic immunosuppression. All patients showed prompt engraftment. Acute GvHD was found in 2 patients (Grade IV n=1, Grade I n=1), chronic GvHD did not occur after a median follow up of 471 days for the surviving patients. Two patients died from treatment-related causes, one from GvHD and one from septicemia. Estimated 2 year survival is 78%. Three patients have relapsed so far at day 128, 192 and 201 post transplant and received donor lymphocyte infusions or chemotherapy. The estimated 2 year disease free survival is 57%. Our results show a good feasibility of an allogeneic stem cell transplantation with reduced intensity conditioning for relapsed Hodgkin’s disease despite extensive pre-treatment and mostly partial remissions at transplant. The rate of acute and chronic GvHD was remarkably low in view of the fact, that 5 of 9 patients had mismatched unrelated donors. In summary allogeneic stem cell transplantation should be further exploited as treatment option for refractory and relapsed Hodgkin’s disease in younger patients.
Late Platelets Recovery after Autografting (ASCT) in Patients with Acute Myeloid Leukemia (AML) Is A Prognostically Favourable Factor for Long-Term Remission
Authors: Carella A. M. , Catania G , Varaldo R , Nati S , Congiu A , Vimercati R , Rossi E , Spriano M
Published: April 2020

Althought favourable results with ASCT in patients with AML in first CR have been reported, there are no definitive data indicating that this approach is superior to chemotherapy alone. We have recently reviewed our 23 years experience with ASCT in AML in Genoa. From January 1984 to December 2007, 79 patients in first CR, aged 18–65 years, who did not have a sibling donor, were autografted. These patients had been previously treated in induction with Ara-C 200 mg/m2/d × 7 days and DNR 50 mg/m2/d × 3 days. Soon after, they had received consolidation therapy with 4 courses of DAT therapy (DNR 50 mg/m2/d1, Ara-C 200 mg/m2 c.i. day 2–5, thioguanine 80 mg/m2 days 2–5). Fifty patients were autografted with bone marrow and 29 patients with peripheral blood stem cells. High-dose therapy consisted of Cyclophosphamide and single dose TBI (n=50) or high-dose busulfan and cyclophosphamide (n=29). At a median of 11 years, 15 patients (19%) are still alive and well. At that time genetic aberrations combined or not with cytogenetics were not available; therefore we have retrospectively analyzed the patients according to age, sex, WBC count, time to CR and conditioning regimen. None of these factors was predictive for long-term remission. On the contrary, we found that late platelets recovery was predictive. The median time to recover platelets > 50 × 109/L was 72 days (45–115 days) in long-term remission patients versus ≤ 30 days in the other patients who relapsed. During the presentation details of this retrospective study will be discussed. In conclusion, the late recovery of platelets after autografting is in our experience the most predictive factor for long-term remission duration in autografted AML patients.
Single Autologous Stem Cell Transplant Using Busulfan and Cyclophosphamide as Conditioning Regimen in Patients with Multiple Myeloma
Authors: Talamo Giampaolo , Claxton David F. , Drabick Joseph , Dougherty David W. , Sivik Jeff , Ehmann Christopher W. , Rybka Witold
Published: April 2020

Autologous peripheral blood stem cell transplantation (ASCT) has been shown to improve survival in patients with multiple myeloma (MM). High-dose melphalan is considered the current standard of care among the preparative regimens used in ASCT for MM patients. We report the results of ASCT in 79 consecutive MM patients using a conditioning regimen with busulfan and cyclophosphamide (Bu/Cy), given as single ASCT and without maintenance therapy. Peripheral blood stem cells were mobilized with cyclophosphamide 5,000 mg/m2 IV + etoposide 1,000 mg/m2 IV, followed by granulocyte colony stimulating factor (G-CSF) 5 mg/Kg/day until the end of stem cell collection. A median of 41.1 × 106 CD34+ cells/Kg (range, 2.1–139.7 × 106) of ideal body weight were mobilized. The conditioning regimen consisted of busulfan 1 mg/Kg PO or 0.8 mg/Kg IV every 6 hours x 16 doses (days -7 to -3), and cyclophosphamide 60 mg/Kg/day IV for 2 days (days -3 to -2). Patients achieved neutrophil engraftment (absolute neutrophil count >500/μL) at a median of +13 days (range, +6 to +21 days), and platelet engraftment (platelets >20,000/μL unsupported by transfusion) at a median of 14 days (range, +11 to +24). Using a transfusion threshold of hemoglobin <8.0 g/dL and platelets <10,000/μL, patients required a median of 2 units of RBC transfusions (range, 0–8), and 1 platelet transfusion (range, 0–15) until hematologic engraftment. Forty-eight and 20 patients reached PR and CR, respectively, for an overall RR of 86%. At a median followup of 41 months (range 2–132 months), the estimated median overall survival (OS) and progression-free survival (PFS) were 45 months [95% confidence interval (CI) = 38–92] and 20 months (95% CI = 15–25), respectively. Veno-occlusive disease developed in 4 pts, and it was lethal in 1 of them. The Bu/Cy regimen was overall well tolerated, and transplant-related mortality was 4%. No statistically significant difference in terms of OS and EFS were observed between the group of patients receiving oral (n=13) vs IV busulfan (n=66). OS was not statistically different between the group receiving ASCT in first remission (n=62) and the group receiving ASCT as salvage therapy, i.e., upon MM progression (n=17), either calculating OS from the day of ASCT or from the day of MM diagnosis. We conclude that our reported clinical outcomes of the Bu/Cy regimen are equivalent to those obtained in historical controls with high-dose melphalan followed by a single ASCT. Thus, given the equivalent effectiveness but greater complexity of administration of the Bu/Cy regimen compared with that of single agent melphalan, we believe the latter should remain the agent of choice for ASCT in MM.
Autologous Hematopoietic Stem Cell Transplantationfor Adult with Acute Leukemia in First Complete Remission
Authors: Zhou Huifen , Depei Wu , Tang Xiaowen , Sun Aining , Jin Zhengming , Qiu Huiying , Miao Miao , Fu Chengcheng , He Guangsheng , Han Yue
Published: April 2020

Objective The objective of this study was to evaluate the clinical outcome of autologous hematopoietic stem cell transplantation (AHSCT) for acute leukemia(AL).

Methods The outcome of 56 AL patients who received autologous HSCT(AHSCT) for AL in first complete remission (CR) from May 2001 to May 2008 were analyzed retrospectively. There were 31 males and 25 females, the median age was 25(11~ 57)years. The diagnosis of these patients were acute myeloid leukemia (AML, n=38), acute lymphocytic leukemia(ALL, n=17) and acute hybrid leukemia (AHL, n=1). Patients were divided into low/moderate risk group (n=46) and high risk group (n=10) according to their characteristics of newly diagnosis. If the patient achieved first complete remission(CR), one to three courses of intensification chemotherapy containing high dose cytarabine(HDAC)were given. All the patients were in CR1 before AHSCT. Stem cells were mobilized with MAG regimen which composed of HDAC, Mitoxantrone and G-CSF. The conditioning regimen consisted of modified BU/ CY for AML patients or total body irradiation (TBI) plus cyclophosphamide (CTX) for ALL patients. Patients received consolidation chemotherapy every 3 months after AHSCT until 3 years post transplant. Nine patients received several course of cytokine-induced killer (CIK) cells treatment.

Result The median number of MNC and CD34 positive cell was 6.9(1.2 `21)×108/kg and 2.3(1.6 `6.3)×106/kg respectively. All patients achieved hemopoietc recovery. The median time of the engraftment of neutrophil and platelet was 9.7(8 `14) days and 12.9(9 `20) days posttransplant respectively. The incidence and severity of regimen-related toxicity were mild. The incidence of treatment related mortality (TRM)is 0%. The median follow-up of surviving patients was 31 months(range, 2 `86). Until now 46 patients are still alive, including 4 relapse cases. 13 cases were died of relapse. The five-year overall survival(OS) and the leukemia-free survival(LFS) were 77.3% and 71.9% respectively. The incidence of relapse was 23.2%. The OS and LFS in the low /moderate risk and high risk AL cohorts were 85.7%versus 40% and 79%versus 40% respectively(P<0.01)

Conclusion AHSCT is the first option for the AL patients who were in good or moderate risk group and had achieved first complete remission. Regular consolidation chemotherapy after transplantation was beneficial to improve the LFS of AL patients.
Expression Profile of Selected HOX Genes in Different Childhood ALL Subtypes
Authors: Vicenova Blanka , Krejci Ondrej , Zaliova Marketa , Muzikova Katerina , Starkova Julia , Pospisek Martin , Drabkin Harry A. , Trka Jan
Published: April 2020

Homeobox-containing (HOX) genes represent a large family of transcription factors that are involved in anteroposterior pattern formation during embryonic organogenesis. Besides their role in ontogenesis, various HOX genes have been associated with haematologic disorders including leukaemia. Multiple studies revealed that dysregulation of HOX genes is relevant to AML, ALL and CML; in addition, HOX genes are rearranged in some cases of human leukaemia and make part of fusion proteins such as NUP98-HOXA9, PBX1-E2A, etc. We have previously studied the pattern of HOX gene expression in chromosomally defined subsets of acute myeloid leukaemia and have shown that HOX genes exhibit a specific expression pattern throughout the AML subsets that can be related to the patient outcome. In this study, we studied the expression profile of various HOX genes in childhood acute lymphoblastic leukaemia (ALL) subtypes. We analysed mRNA levels of 23 different homeobox-containing genes in 59 children patients with ALL that were divided into six groups based on cytogenetic abnormalities and prednisone responsiveness: BCR/ABL, hyperdiploid ALL, TEL/AML1, MLL/AF4, T-ALL, prednisone good responders (PGR) and prednisone poor responders (PPR). Patient cDNA samples were analyzed using SYBRGreen I real-time quantitative PCR (qRT-PCR) and data obtained were normalized to the expression of ABL control gene. Our data indicate that certain HOX genes including HOXA3, HOXA4, HOXA5, HOXA6, HOXA9, HOXA10, HOXB2 or HOXB3 are differentially expressed throughout the ALL subtypes. Most notably, HOXA4, HOXA9, HOXB2 and HOXB3 mRNA levels appear to be elevated in T-ALL patients; hyperdiploid ALL patients display increased HOXA5 mRNA levels and HOXA3, HOXA6, HOXA9 and HOXA10 expression is higher in patients bearing the MLL/AF4 fusion gene. A relatively high level of HOXA5, HOXB2 and HOXB3 expression was observed in all patient groups. Furthermore, HOX gene expression distinguishes PGR from PPR group of patients; this divergence is most apparent in the case of HOXB2, HOXB3 and HOXB7. This study shows potential prognostic relevance of HOX gene levels in patients with childhood ALL. Data also confirm the differential HOX gene expression in various childhood ALL subtypes and underline the importance of certain HOX genes in leukemogenesis.
Molecular Variability of FLT3/ITD Mutants and Their Impact on the Differentiation Program of 32D Cells: Implications for the Biological Properties of AML Blasts
Authors: Pekova Sona , Dvorak Michal , Ivanek Robert , Rueggeberg Sabrina , Leicht Stefan , Kozak Tomas , Vrba Jiri , Schwarz Jiri , Cetkovsky Petr , Prucha Miroslav , Hrusak Ondrej
Published: April 2020

FLT3 is the most frequently mutated gene in AML – up to 40% of AML harbor an activating mutation within FLT3 gene. Though AML is a relatively rare disease, such a high mutability rate, as observed with FLT3 gene, is striking. To elucidate the molecular background of this phenomenon, we have established nine unique FLT3/ITD - carrying 32D cell lines and a set of controls, and subjected them to whole genome expression analysis and 2DE LC/MS proteomics. Data obtained on this so far largest set of ITD mutants indicates that FLT3/ITD positive cells exhibit less mature expression profiles resembling ST-HSC and MkEP/CMP/LMPP progenitors. FLT3/ITD-positive 32D cells overexpress a combination of genes of pluripotency (Etv5, endoglin, EpoR, Mpl, Trfr2, GATA.2, Hemgn, Myb, Dok2, Gli1, Pim3, PML, Ikaros, Pum1, Dlx4, Hox-c8, HoxB9, Runx1, c-Myc, Shh, FL) and early erythroid precursors (EpoR, Trfr2, GATA.1, Klf1), implicating their less mature expression profile than 32D cell lines harboring wild type FLT3 or 32D controls. We hypothesize that FLT3/ITD might contribute not only to the proliferative advantage of FLT3/ITD positive cells, but also to their reprogramming towards less differentiated stages, thus strengthening their malignant properties. This finding might explain the pronounced mutation rate of the aberrantly expressed FLT3 gene in AML, and, also, the inferior prognosis of FLT3/ITD positive AML patients. Moreover, the microarray data has revealed biological differences among individual ITD variants – a finding supporting the recent clinical data on the prognostic impact of the size of individual ITDs.
Factor V Deficiency in a Patient with Polycythemia Vera and Huntington’s Chorea
Authors: Girtovitis Fotios I , Diamantidis Michael D. , Kaiafa Georgia , Constantinou Nikolaos , Lazaridou Despina , Saouli Zoi , Ntaios George , Kontoninas Zisis , Andreadis Charalambos , Papadopoulos Athanasios
Published: April 2020

Factor V deficiency is a very rare congenital disorder of blood coagulation, which was initially described by Owren in 1947. Ever since it is estimated that approximately 150 cases have been reported worldwide. We describe a case of a 31-year-old female, who was referred to the hemostasis unit of our clinic for investigation of a prolonged prothrombin time (PT) [PT: 14.8″ (control 12″)], along with a prolonged activated partial thromboplastin time (aPTT) [aPTT: 41.9″ (control 31.5″)]. These disorders were discovered in a pre-operating check-up, before being subjected to a removal of a thyroid gland node. Her medical history revealed that she had been diagnosed with polycythemia vera 6 years before, as well as Huntington’s chorea almost a decade earlier. The patient was under phlebotomy therapy once or twice a month, with no other specific treatment. She received acetylsalicylic acid (100mg/day), because of polycythemia vera, which had to be discontinued, due to mild ecchymoses and menometrorrhagia. Apart from the bleeding tendency mentioned above (attributed to administration of acetylsalicylic acid), no other hemorrhagic manifestations arose from her whole medical history. Her hematological profile was as follows: WBC: 7500/μl, Hb: 14,4 g/l, PLT: 710.000/μl. The patient’s hepatic and renal examination was normal. Repeat PT and aPTT after mixing with normal plasma gave normal results, thereby excluding the presence of a coagulation inhibitor and indicating deficiency of a factor in the common pathway. Factor V assay was done and it was found to be 15%. The examination of the rest coagulation factors was within normal range. The possible association of such a coagulation disorder with the aforementioned diseases has not been established until today. We therefore consider interesting the case described, not only because of its rarity, but also because of its underlying pathology and concomitant disease.
Thromboelastogram Platelet Mapping Accurately Predicts Bleeding Phenotype in Glanzmann Thrombasthenia
Authors: Callaghan Michael U , Chitlur Meera B. , Lusher Jeanne M. , Rajpurkar Madhvi , Fleming Patricia
Published: April 2020

Glanzmann thrombasthenia (GT) is caused by genetic defects in the ITGA2B and ITGB3 genes that encode the proteins of the αIIbβ3 complex on the surface of the platelet. The αIIbβ3 complex on activated platelets acts a receptor principally for fibrinogen but can also bind fibronectin, vonWillebrand factor, vitronectin and CD40 ligand under conditions of high flow. The binding of these proteins results in the formation of protein bridges and aggregation of platelets. Patients with GT have deficiency of platelet surface αIIbβ3 resulting in insufficient platelet spreading and aggregation and resultant bleeding. Conventional platelet aggregation studies demonstrate aggregation with ristocetin but absence of aggregation with other platelet agonists including thrombin, collagen, arachadonic acid and epinephrine. Platelet immunophenotyping demonstrates absent (Type I) or diminished (Type II) surface expression of CD41 (Gp IIb) and CD61 (Gp IIIa). Bleeding phenotype in individual patients can be quite variable and is not well predicted by surface expression or platelet aggregation studies.

Thromboelastogram platelet mapping (TEG-PM) is a newer technology that employs antagonists of coagulation cascade meditated thrombosis in concert with platelet agonists to determine the level of platelet inhibition. Here we have examined the bleeding phenotype (Table I) of 5 GT patients, from three separate families, followed at our center in relation to conventional platelet aggregation studies, platelet immunophenotype and TEG-PM (Figure I). Three of the patients are from a single kindred and have had frequent severe bleeding episodes requiring treatment with activated FVII, transfused platelets and transfusion support for blood loss. The fourth patient has had an intermediate bleeding phenotype. The fifth patient has had only a single severe bleeding episode after nasal surgery and an otherwise mild bleeding phenotype including a caesarian section with a single platelet transfusion and no bleeding. The fifth patient displayed reduced CD41 and CD61 platelet expression (type II GT) while the other 4 patients have no CD41 and CD61 platelet expression (type I GT) by flow cytometry. While all 5 patients have similar, classic platelet aggregation studies the TEG-PM accurately predicted the bleeding phenotype. We believe TEG-PM may be a useful tool for the management of patients with GT.

Table I

Patient I Patient II Patient III Patient IV Patient V Age 4 years 10 years 20 years 2 years 29 years Sex Female Female Male Female Female # of PRBC Transfusion 0 2 0 3 0 Hospitilizations 2 4 7 4 1 FVIIa days 69 47 27 17 0 Platelet transfusions 9 3 13 0 1 PFA Epi >262 s - - >196 s 259 s PFA ADP 272 s - - >210 s 173 s TEG K - - - 7.8 min 2.2 min TEG MA 13.6 13.4 16.4 22.8 65.9 Platelet Count 83–439K 91–364K 76–449K TEG ADP Inh 89.3% 100% 83.2% 100% 6.8% TEG AA Inh 98.7% 93.2% 89.7% 100% 68.6%

Figure I Figure I.
Spontaneous Resolution of Extreme Essential Thrombocytosis in a Child
Authors: Aviner Shraga , Schlesinger Menachem , Lugassy Gilles
Published: April 2020

Essential Thrombocytosis (ET) is rare in children and its clinical course is considered to be different from the adult disease. Unlike adults, most children with ET do not need treatment; however, children with extreme thrombocytosis present the treating physician with a therapeutic dilemma. We report a girl with a spontaneous resolution of extreme essential thrombocytosis. A 13 year old previously healthy girl presented to our ER with a 3 months history of intermittent mild abdominal pain with short episodes of diarrhea, intermittent episodes of paraesthesia in lower limbs and 3000 × 109/L platelets on CBC with pseudohyperkalemia of 6.4 mEq/L. Physical examination was unremarkable except for some diffuse mild tenderness on abdominal palpation. Laboratory work-up revealed normal WBC and Hb, Platelets 4100 × 109/L, mild microcytosis, Iron - 21.9 mcg/dL, transferin saturation 5.6%, serum potassium − 6.5 mEq/L, LDH 1000 IU. Liver, renal and thyroid function tests were within normal limits. PTT was mildly elevated 49.3 seconds (normal – 24.3–35), LAC-R mildly positive – 1.205 (0.8–1.20), KCT 58.2 sec. Anticardiolipin IgG −4.82 U/ml (negative), IgM - 11.24 U/ml (negative < 7 U/ml). Anti dsDNA and Anti nuclear antibodies were negative. Studies for hypercoagulable state included Protein C, Antitrombin, Factor VIII, IX, XI, Factor V R506Q, and Apolipoprotein a; all were normal. A heterozygosity for PT G20210A was found, as well as homozygosity for MTHFR C667T with a normal level of Homocysteine (7.7mmole/L). Bone marrow aspiration showed increased cellularity of all cell lines, with many young megakaryocytes including some in clusters. No malignant cells were seen. Reticulin score on bone marrow biopsy was 0. Platelets function tests with adenosine di-phosphate and epinephrine were normal. Serum TPO level was 31.0 pg/ml (normal). A karyotype examination revealed a normal 46XX female. FISH for BCR/ABL was negative. DNA mutations for JAK2 on both the 12 and the14 exons and c-mpl mutations were not found. Spontaneous clone formation was normal. Helicobacter pylori IgG was 2.89 U/ml (normal) and stool was negative for parasites. Imaging studies included high resolution abdominal contrast CT scan, upper gastrointestinal tract imaging with gastrografin, Doppler sonography of splanchnic vessels and capsule endoscopy; all were interpreted as normal. Aspirin 75 mg/d was started without antimetabolite therapy. Abdominal pain and diarrhea gradually improved. After 3 month of antiagregant therapy, platelets count declined, gradually but steadily. One year after presentation the child is asymptomatic with platelet count of 600 × 109/L. The spontaneous remission of the extreme ET achieved by conservative treatment with no complications adds to the growing evidence, that childhood ET is a unique entity with different biology than adult ET, which might not need antimetabolite therapy.
Real-World Clinical Experience with Long-Term Miglustat Therapy in Type 1 Gaucher Disease: The ZAGAL Project
Authors: Giraldo Pilar , Alfonso Pilar , Fernandez-Galan MAngeles , Martín-Nuñez Guillermo , Barez Abelardo , Franco Rafael , Alonso Dora , Acedo Antonio , Martin Alejandro , Martinez-Estefano Elvira , Roldan Vanessa , Serrano Sol , Latre Paz , Pocovi Miguel
Published: April 2020

Background and objectives: Several reports describe the use of miglustat (Zavesca®), an oral inhibitor of glucosylceramide synthase, for type 1 Gaucher disease (GD1) in clinical trials. There are few data on the use of miglustat for GD1 in the real-world clinical experience. Here, we assess the long-term efficacy, tolerability and safety of miglustat in a prospective, open-label, observational study of GD1 patients attending routine clinic visits.

Design and Methods: 48 patients with mild-to-moderate GD1 received miglustat 100 mg t.i.d. over 36 months. Patients were analysed in two groups: therapy-naïve patients (n = 11) and those switched from previous treatment with enzyme replacement therapy (ERT; n = 37). Assessments of clinical status, haematological parameters, and organomegaly were performed before treatment and after 6, 12, 24 and 36 months of therapy. Data were compared with a historical reference cohort 29 GD1 patients treated for 36 months with ERT.

Results: Nine treatment-naïve patients completed 36 months of miglustat therapy. Sustained improvements in haemoglobin were seen in all patients who had anaemia before treatment. Platelet counts improved in patients with initial thrombocytopenia, and were maintained in patients with normal counts at baseline. Plasma chitotriosidase activity decreased in treatment-naïve patients during the first year on therapy and remained stable thereafter. S-MRI findings indicated improved bone status at 36 months. In patients switched from ERT, 15 completed 36 months of miglustat therapy, and displayed maintained or improved clinical and haematological parameters, and surrogate disease severity markers. No statistically significant differences were observed at 36 months between treatmentnaïve patients with similar baseline characteristics treated with either miglustat or ERT. Overall, miglustat was well tolerated.

Interpretation and conclusions: Miglustat provided appreciable therapeutic responses on clinical, haematological, biomarker and S-MRI parameters at 36 months in patients with GD1, and was well tolerated. Therapeutic responses with miglustat were comparable with those seen with ERT.