bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Hypusination of Eukaryotic Initiation Factor 5a (eIF-5A) as a Novel a Synergistic Treatment Strategy in Imatinib-Treated BCR-ABL Positive Leukemias Identified by a Global Proteomics Approach.
Authors: Balabanov Stefan , Ziegler Patrick , Hartmann Ulricke , Kammer Winfried , Copland Mhairi , Priemer Martin , Wilhelm Thomas , Bokemeyer Carsten , Kanz Lothar , Holyoake Tessa , Nordheim Alfred , Bruemmendorf Tim
Published: October 2019

Imatinib (IM) is highly effective in the treatment of CML and remains the gold standard non-transplant therapeutic option. However, resistance to IM develops frequently, particularly in late stage disease. The aim of this study was to discover novel cellular Bcr-Abl targets leading to the identification of potential novel treatment options that would synergise with IM. Protein expression analysis of K562 leukemic cells after IM treatment was performed using 2-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF) and nano electrospray ionization tandem mass spectrometry (ESI-MS/MS). Differential protein expression of selected proteins was confirmed by Western blot analysis. Among others, we detected the IM-dependent down-regulation of the eukaryotic translation initiation factor 5A (eIF-5A), which is essential for cell proliferation. EIF-5a represents the only known eukaryotic protein activated by a unique, 2-step post-translational modification called hypusination, a process that can be specifically inhibited by hypusination inhibitors (HI), e.g. ciclopirox and GC7. We demonstrated that selective HI elicit a strong synergistic effect with IM on both cellular cytotoxicity (using MTT assay) and apoptosis (using PI staining) of Bcr-Abl positive leukemic cell lines and even more strikingly on primary CD34+ cells from CML patients at diagnosis. The effect of HI on CD34+ cells from healthy donors and CML patients was compared by short-term expansion in serum free medium using high resolution cell cycle analysis and carboxy-fluorescein succinimidyl ester (CFSE) labelling to track cell division. We observed a substantial anti-proliferative effect of the drug combination on Bcr-Abl positive as compared to Bcr-Abl negative cells. However, we were unable to demonstrate a significant cytotoxic effect of the combination on the quiescent Ph+ stem cell population. Despite this, HI exerted strong antiprolifertive effects not only on Ba/Fp210 but also on resistant Ba/F3p210 mutants T315I, E255V and M351T.

In conclusion, IM induced significant changes of global cellular protein expression in K562 cells, including down-regulation of eIF-5A. Inhibition of eIF-5A hypusination represents a promising target for combined therapy in Bcr-Abl positive leukemias. Ongoing studies are investigating whether novel, more specific, HI in combination with IM (and/or 2nd generation Bcr-Abl inihibitors e.g AMN107, BMS-354825) will be able to exert these effects on the quiescent Bcr-Abl+ stem cell compartment, and if these novel combinations are effective in IM-resistant cells.
Phosphorylation Levels of BCR-ABL, Stat5, and CrkL Proteins in Imatinib-Resistant CML Patients as Compared to Previously Untreated CML Patients: Implication of Poor Drug Binding and Activation of Alternate Pathways.
Authors: Gorre M. , Kantarjian H. , Jilani I. , Cortes J. , Salvado A. , Rae P. , Alland L. , Dugan M. , Ottmann O. , Bhalla K. , Giles F. , Albitar M.
Published: October 2019

Disease progression in patients with chronic myeloid leukemia (CML) while on imatinib therapy has been reported, and ex-vivo studies have suggested that this resistance can be seen despite adequate suppression and deactivation of the BCR-ABL fusion protein (Donato et al. Blood. 2003;15:690). Whether the levels of BCR-ABL phosphorylation differ between imatinib-resistant and imatinib-sensitive patients is not known. We compared the relative phosphorylation of BCR-ABL protein (ratios of phospho-tyr245-BCR-Abl:total BCR-ABL protein and phospho-thr735-BCR-ABL:total BCR-ABL protein) in 54 previously untreated CML patients and 62 CML patients with progressive disease who were resistant to imatinib. Resistant patients had significantly lower levels of phospho-thr735-BCR-ABL protein than previously untreated patients, with a median ratio of 0.64 (range, 0.03–1.63) vs. 0.80 (range, 0.40–1.67) (Wilcoxon test, P <0.001). Ratios of phospho-tyr245-BCR-ABL were also lower in patients with resistant progressive disease, with a median of 0.71 (range, 0.02–2.18) vs. 0.80 (range, 0.30–2.15) in previously untreated patients (P=0.03). Furthermore, when quantitative flow cytometry (QuantiBRITE) was used to measure CrkL phosphorylation, resistant patients had significantly lower levels of phosphorylated CrkL/cell than did previously untreated patients (median of 4204 mol/cell Vs. 5982 mol/cell; P=0.01), despite an increase in the overall percentage of p-CrkL-positive cells due to an increase in blasts. Similarly, the levels of AKT phosphorylation as measured by quantitative flow cytometry showed significantly lower intensity p-AKT in resistant patients than in previously untreated patients (median of 4131 mol/cell vs. 5949 mol/cell; P=0.008), again despite an increased percentage of p-AKT-positive cells due to an increase in blasts. There was no significant difference in Stat5 phosphorylation as measured by quantitative intensity, but the percentage of p-Stat5-positive cells was significantly higher in resistant patients than in previously untreated patients (median 22% vs. 8%; P=0.02). This data confirms that in imatinib-resistant CML patients, the disease resistance is not necessarily dependent on higher activity in the BCR-ABL and AKT pathways, but is more likely due to inability of imatinib to bind and suppress BCR-ABL (possibly due to mutations) or to the activation of other pathways that render the disease resistant. Further studies are needed to focus on specific mutations and the activation of other pathways.
Heat Shock Protein 70 over Expression Is Associated to Imatinib Resistance in Chronic Myelogenous Leukemia.
Authors: Pocaly Marion , Lagarde Valérie , Etienne Gabriel , Ribeil Jean-Antoine , Bonneu Marc , Claverol Stéphane , Moreau-Gaudry François , Hermine Olivier , Turcq Béatrice , Mahon François-Xavier , Pasquet Jean-Max
Published: October 2019

Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative syndrome characterised by the expression of the recombinant oncoprotein Bcr-Abl. Imatinib inhibits Bcr-Abl tyrosine kinase activity leading to apoptosis of leukemic cells sparing normal hematopoiesis. Several mechanisms of resistance to imatinib have been identified both in vitro and in vivo: Bcr-Abl mutations, an over-expression of the Bcr-Abl kinase itself or other tyrosine kinase bypass. To identify unknown mechanism, we used an imatinib resistant cell line (K562-R) generated from the erythroblastic cell line K562 (K562-S) (Blood, 2000; 93: 1070–1079) for which all described mechanisms of resistance have been previously invalidated. Previous results from a proteomic study identified some chaperon proteins such as heat shock proteins with an increased expression level in K562-R. One of them, the heat shock protein 70, Hsp70, has a 3 fold increase expression level in K562-R cells, results which have been confirmed by western-blot analysis. To characterise the role of Hsp 70 in imatinib resistance, we inhibit Hsp 70 expression by RNA silencing (siRNA) in K562-R cells and over-express it in K562-S cells. Inhibition of Hsp70 protein expression by siRNA decrease Hsp70 expression rapidly over 90% at day 4 which is associated with a significant reduction of viability (66 ± 6%, n = 5, p < 0.03). Over expression of Hsp 70 in K562-S cells induced a significant increase of resistance to imatinib since the addition of imatinib only increases mortality by 27 ± 5 % in comparison to 52 ± 4 % for K562-S cells (n = 4, p < 0.001). Detection of HSF-1 phosphorylation, the major transcription factor involved in Hsp 70 expression, did not show significant differences between K562-S and K562-R cells although over a 3 fold increase is detected in the mRNA level of Hsp 70 in K562-R cells by quantitative PCR. Furthermore, the comparison of Hsp70 expression in mononuclear cells of 7 CML patients before imatinib treatment and at the relapse time shows that Hsp 70 is increased in imatinib resistant patients suggesting it could also play a role in resistance in vivo. Present study confirmed that over expression of Hsp 70 in the cell line K562-R is involved in the mechanism of imatinib resistance in vitro. Moreover, the correlation between the increase of Hsp 70 in CML patient cells and resistance suggests it could be an interesting marker and potentially a therapeutic target.
Adoptive Immunotherapy with Donor Lymphocyte Infusions after T-Cell Depleted Allogeneic Stem Cell Transplantation for Patients with Acute Myeloid Leukemia.
Authors: Barge Renee M.Y. , Steel Eva , Starrenburg Ingrid , Beaumont Floor , Smit Willem M. , Schaafsma Martyn Ronald , Posthuma Ward , Fibbe Willem E. , Falkenburg Frederik , Willemze Roelof
Published: October 2019

In this study we analyzed 40 patients with AML who received donor lymphocyte infusions (DLI) after allogeneic stem cell transplantation (alloSCT). Patients with a median age of 42 (range, 11–66) were transplanted with an in vitro T-cell depleted (Campath in the bag) graft from an HLA matched sibling following a myeloablative (n=35) or a nonmyeloablative conditioning regimen (n=5). Patients received DLI for relapsed AML (n=28) or for mixed chimerism (n=12). All patients with relapsed AML were treated with a cytarabine-containing chemotherapy regimen or with gemtuzumab ozogamycin immediately prior to DLI treatment and received daily 3 x 106 U α-IFN sc. The DLI dose administered was depending on the protocol used. Fourteen relapsed patients received high dose DLI with a median of 50 x 106 CD3+ cells/kg and 14 low dose DLI with a median of 5 x 106 CD3+ cells/kg. Ten of 28 patients with relapsed AML obtained a complete remission after the combination of chemotherapy and DLI. One patient obtained a partial remission and fourteen patients showed no response. Three patients were not evaluable. No effect of cell dose was observed on the clinical response after DLI. The median percentage of donor chimerism in patients with relapsed AML before DLI was 74% (range, 5–96%) and increased after DLI to 97% (range, 5–100%). Six patients with relapsed AML became complete chimera after DLI. DLI caused in 12 of 28 relapsed patients grade 3–4 GVHD. cGVHD was observed in 3 patients of whom 2 developed extensive GVHD. With a median follow up of 11 months only 1 pt in the high dose and 4 pts in the low dose group are alive in CR and 23 have died due to progressive disease (16) or GVHD (7).

Due to this low rate of long-term survival of relapsed AML patients treated with DLI, we recently developed a new DLI protocol for AML patients. Twelve patients with mixed chimerism at 6 months after T-cell depleted alloSCT were pre-emptively treated with 3 x 106 CD3+ cells/kg. Complete chimeric response was observed in 6 of these 12 patients. Two patients showed increasing mixed chimerism and three patients showed no response. Two of 12 patients treated for mixed chimerism developed aGVHD gr 1–2. No severe aGVHD was observed in this group. Two patients developed chronic GVHD, both after nonmyeloablative conditioning. Incidence of GVHD was significantly lower in this group compared to the relapsed patients. Currently, 7 patients are alive in CR. In conclusion: 1) Long term survival of patients with overt relapse AML after alloSCT treated with DLI remains poor 2) High dose DLI appeared to be less effective than low dose DLI due to increased toxicity 3) DLI for AML may be more successful when it is administered pre-emptively for mixed chimerism after T-cell depleted alloSCT.
Serum Free Light Chain [FLC] Assay in Multiple Myeloma Patients Who Achieved Negative Immunofixation after Allogeneic Stem Cell Transplantation.
Authors: Moesbauer Ulrike , Schieder Heike , Renges Helmut , Ayuk Francis , Zander Axel , Kröger Nicolaus
Published: October 2019

The quantitative assay for free light chains [FLC] has been reported to be sensitive and specific for detecting and monitoring free light chain diseases such as multiple myeloma. To evaluate the sensitivity of FLC for monitoring patients in complete remission for early detection of relapse, the measurement of more than 250 serum free light chains were performed with the commercial available Freelite TM kit [Binding Site] in 26 patients who achieved complete remission with negative immunofixation after dose reduced allogeneic stem cell transplantation. The patient groups were divided in those who remained immunofixation negative [n=12, group 1] during follow-up of at least 1 year and those who had been immunofixation negative but became positive during follow-up [n=9, group 2] and those who had achieved near complete remission with positive immunofixation but then became immunofixation negative during follow-up [n=5, group 3]. In group 1 the measuring of 105 FLC concentration and kappa/lambda ratio was performed in 12 patients. In 10 patients [83 %] free light concentration of kappa or lambda remained within the normal range during follow-up of more than 1 year. In 2 patients [17 %] kappa or lambda FLC concentration was above the normal range, but remained stable without any signs of increasing amount. Group 2 consisted of 9 patients who had been immunofixation negative but became positive during follow-up. In all patients an increase of the corresponding free light chain could be observed in serum. In 4 patients a very close monitoring of immunofixation and free light assay was performed and an at least 25 % increase of the free light concentration in serum was observed at a median of 97 days before immunfixation became positive. In group 3 five patients who had been immunofixation positive became negative during follow-up. In all of the patients the free light concentration was within the range at time of negative immunofixation. The corresponding free light concentration dropped down and reached normal level at a median of 38 days before the patients had achieved negativity of immunofixation. These results suggest that serum free light chain assay allows monitoring of patients with complete remission and might detect early relapse before immunofixation becomes positive. Thus, an early increase of free light chain assay in immunofixation negative patients after allogeneic transplantation might be an useful guide for adoptive immunotherapy strategies to prevent clinical relapse.
Outcome of Allogeneic Stem Cell Transplantation for Patients with Severe Aplastic Anemia after Conditioning with Procarbazine, ATG and Cyclophosphamide: A Single Center Experience.
Authors: Seong Ki , Lee Jong Wook , Kim Yoo Jin , Kim Hee Je , Ki Chang , Lee Seok , Yong Sung , Sung Woo , Choo Kim Chun
Published: October 2019

Background: Cyclophosphamide (CY, 200 mg/kg) plus ATG seems to be accepted as a standard conditioning regimen in HLA-matched sibling stem cell transplantation (SCT) for severe aplastic anemia (SAA). We report herein the outcome of allogeneic SCT for the patients with SAA conditioned with procarbazine (PCB), ATG and CY.

Methods: Between January 1995 and March 2004, consecutive one hundred and sixty two patients with SAA received matched sibling SCT after conditioning with PCB, ATG and CY. The median age of patients was 28.5 (16~50) and median interval between diagnosis and SCT was 10.5 months (1~216). Fifty-five patients (34%) had a history of immunosuppressive therapy before SCT. The conditioning regimen consisted of PCB (6.25 mg/kg/day, 6 days), CY (50 mg/kg/day, 4 days) and ATG (1.25 mg/kg/day, 3 days). No graft manipulation was performed in 92 patients (52%), and donor marrow expansion and megadose transplantation (bone marrow plus CD34+ isolated PB as a stem cell source) were done in 35 patients (21.6%). All patients received of cyclosporine and methotrexate as GVHD prophylaxis.

Results: The median dose of CD34+ and CD3+ cells infused were 4.8 x 106/kg (0.8~49.6) and 5.1 x 107/kg (0 ~ 38), respectively. Median time to neutrophil recovery more than 500/μL and platelet recovery more than 20,000/μL were 13 days (0~49) and 19 days (0~150), respectively. All patients achieved successful primary engraftment, but delayed engraftment failure developed in 18 patients (11%). Ten among 18 patients who developed graft failure received second SCT, resulting survival of 9 patients. The incidence of acute (more than grade II) and chronic GVHD were 9.3% (n=15) and 13.0 % (n=21), respectively. DFS and overall survival (OS) at 6 years was also 90%. Factors influencing on DFS on univariate analysis were patient and donor’s age, development of acute and chronic GVHD, and times of pregnancy (over 2 times). Development of acute and chronic GVHD, and occurrence of rejection were factors that influence DFS on multivariate analysis. Causes of death (n=15) were pneumonia (n=4), rejection (n=4), GVHD±infection (n=2), chronic GVHD (n=1), MOF (n=1), CVA (n=1), autoimmune hemolytic anemia (n=1), and unknown (n=1).

Conclusions: These data suggest that the conditioning regimen used in this study is feasible for patients with SAA who received matched sibling SCT. GVHD and rejection were the most important factors that influence DFS.
Fludarabine Based Conditioning for Allogeneic Transplantation in Severe Aplastic Anemia.
Authors: Chandy Mammen , George Biju , Viswabandya Auro , Mathews Vikram , Bajel Ashish , Srivastava Alok
Published: October 2019

Patients with severe aplastic anemia (SAA) who are multiply transfused or septic have a poor outcome after allogeneic stem cell transplantation. Forty three patients (31 males and 12 females) with SAA underwent allogeneic BMT using a fludarabine based conditioning regimen between 1998 and 2005. The median age was 20 years (range: 4–38) with 11 children and 32 adults. All donors were 6 antigen matched HLA identical sibling or family donors. Co morbidities seen included bacterial sepsis in 15 patients, fungal pneumonia in 4 and a recent intracranial bleed in 5 patients. Seven patients had failed Antithymocyte or antilymphocyte globulin (ATG/ALG) and two patients had failed their first transplant. The median time from diagnosis to transplant was 12 months (range: 2 – 96) and the median transfusions prior to BMT was 28 (range: 2 – 380). Conditioning therapy consisted of: Fludarabine (Flu) 180 mg/m2 over 6d + Busulfan (Bu) 8 mg/kg over 2d + ATG 40 mg/kg/day over 4 d in 17 patients, Flu 180 mg/m2 over 6d + Cyclophosphamide (Cy) 120 mg/kg over 2d ± ATG 40 mg/kg/day over 4d in 17 patients, Flu/TBI/OKT3 in 4, and Cy 120 + Flu 150mg/m2 in 5 patients. Graft versus host disease (GVHD) prophylaxis consisted of Cyclosporine and mini methotrexate. Graft source was peripheral blood stem cells in 39 patients and G-CSF stimulated bone marrow in 4. The median cell dose was 5.2 x 108 MNC/kg (range: 2.1 – 13.6) for PBSC and 5.2 x 108 TNC/kg (range: 3.7 – 6.8) for bone marrow. Five patients expired within the first 10 days due to sepsis. Thirty seven patients engrafted with a mean time to ANC > 500 of 11.6 days (range: 8 – 18) and median platelet engraftment time of 13 days (range: 8 – 32). One patient had primary graft failure and expired on day 64 due to fungal pneumonia despite a second transplant. Acute GVHD was seen in 14 patients (38%) with Grade III–IV GVHD in 4 (10.5%). Chronic GVHD was seen in 10 patients with 6 having limited and 4 with extensive GVHD. Two patients had secondary graft rejection on day + 24 and +60 respectively and expired due to fungal pneumonia. At a median follow up of 17 months (range: 5 – 78); 29 patients (67.7%) are alive and well. Among patients treated with Flu/Bu/ATG, 12/17 (70.5%) are alive and well while the DFS is 82% (14/17) in patients treated with Flu/Cy ± ATG. Comparison with patients conditioned with Cy/ATG during 1990–2004 is given in the table. This comparison suggests that a fludarabine based conditioning regimen may be better, with less rejection, than Cy/ATG for allogeneic BMT in sick patients with SAA who are infected and multiply transfused at the time of BMT.

Comparative data Fludarabine Cy/ATG Number 43 26 Rejection 3 (7%) 7 (27%) DFS 29(67.7%) 11 (46%)
Birth Order and Outcome in HLA-Identical Sibling Donor Hematopoietic Stem Cell Transplants. Impact of a Sequential Fetomaternal - Maternofetal Cell Transfer?.
Authors: Bucher Christoph M. , Heim Dominik , Buser Andreas , Passweg Jakob R. , Gratwohl Alois
Published: October 2019

Fetomaternal and maternofetal cell transfer have been described. Their clinical relevance is unknown. We hypothesized that firstborn siblings could tolerize their siblings born thereafter through sequential fetomaternal-maternofetal cell transfer. Hence, stem cell transplants within a family from a firstborn sibling (group A) should result more graft-versus-host disease (GvHD) and worse overall survival than transplants to a firstborn donor (group B). Results of a retrospective single center cohort analysis of 321 HLA-identical sibling donor hemopoietic stem cell transplants showed a survival of 48.3% (+/−10.9%) at 10 years in the group with firstborn donors (group A, 110 patients) as compared to 63.2 (+/−10.6%) in the group with firstborn recipients (group B, 105 patients; p<0.02) and a RR of death after adjustment for other risk factors of 2.6 (CI 1.45–4.66; p<0.001) for the group with firstborn donors. These results support the concept of a clinically relevant tolerizing effect of birth order, possibly mediated by fetomaternal cell transfer in man.

Patients characteristics and outcomes Birth Order Donor First Sibling Recipient First Sibling n 110 105 Donor Age median (range) 30.1 (4.9–68) 25.4 (0.4–59.1) p=0.005 Recipient Age median (range) 25.4(3.2–62.1) 30.8 (2.2–63.0) Number of Siblings 2 67 69 n.s. 3 26 22 >3 17 14 Diagnosis n.s. Acute myeloid leukemia n 33 26 Acute lymphoblastic leukemia n 24 24 Chronic myeloid leukemia n 23 23 Lymphoproliferative disorders n 14 13 Severe aplastic anemia n 11 12 Myelodysplastic syndromes n 5 7 Stem Cell Source n.s. Bone Marrow n 71 73 PBSCT n 39 32 Conditioning n.s. With totoal body irradiation n 18 22 Without total body irradiation n 92 83 Outcomes Survival at 10 yrs % 48.3 63.2 p<0.02 Relapse at 3 yrs. % 26 20 n.s. Acute GvHD p=0.017 < Grade II n 46 61 >= Grade II n 64 44 Chronic GvHD n.s. none n 28 30 n.a. n 29 17 Limited n 32 30 Extensive n 21 23 Figure 1: Kaplan Meyer estimate of cumulative survival of groups A(firstborn donor: gray line) and B (firstborn recipient: black line). + indicates censored patient. Figure 1:. Kaplan Meyer estimate of cumulative survival of groups A(firstborn donor: gray line) and B (firstborn recipient: black line). + indicates censored patient.
Double Unit Cord Blood Transplantation in Children with Acute Leukemia.
Authors: Yoo Keon Hee , Kang Hyoung Jin , Lee Soo Hyun , Jung Hye Lim , Sung Ki Woong , Koo Hong Hoe , Shin Hee Young , Ahn Hyo Seop
Published: October 2019

Cell dose is one of the most important factors predicting successful outcome after cord blood transplantation (CBT), and the University of Minnesota group recently reported results of transplantation using 2 partially HLA-matched cord blood units for adult patients. Although a significant proportion of pediatric recipients would benefit by cell dose augmentation, data concerning multiple unit CBT in children are lacking by far. We investigated the feasibility of double unit CBT for pediatric recipients. If the cryopreserved nucleated cell dose of the largest available unit was 3.5x107 or less per kg recipient body weight, double unit CBT were scheduled. Twelve patients with acute leukemia (7 myeloid, 3 lymphoblastic, 2 mixed) aged 6.2–16.9 y (median 13.3 y) received 2 cord blood units at our two cooperative institutions. All patients except one were in complete remission before transplant (4 CR1, 6 CR2, 1 CR3, 1 in 3rd relapse). Patients were conditioned with intravenous busulfan- or TBI-based myeloablative regimens. All transplanted units were 4–6/6 and 3–6/6 HLA-matched to the recipient and to the other co-transplanted unit, respectively. Cyclosporine was used for GVHD prophylaxis with combined use of short-course methotrexate (n=3), methyl-prednisolone (n=4), or mycophenolate mofetil (n=5). Median infused nucleated cell dose was 3.13x107/kg (range, 0.72–6.2x107/kg). All patients engrafted at a median of 23 days (range, 15–34 days). All of the 9 patients who were followed-up longer than 90 days achieved platelet engraftment at a median of 47 days (range, 24–81 days). The initial chimerism analysis performed at 1 mo revealed donor-derived hematopoiesis in all patients including 5 patients with 2 donor mixed chimerism, and one patient showed persistent 2 donor mixed chimerism beyond 1 year following transplant. Grade 2–4 and grade 3–4 acute GVHD were developed in 8 (66.7%) and 2 (16.7%) patients, respectively. Three out of the 9 evaluable patients developed limited chronic GVHD. There were 3 events (2 transplant-related deaths and 1 relapse) and estimated disease-free survival was 70.7% at 18 mo. We conclude that double unit CBT is also safe and feasible in pediatric recipients without a single unit containing adequate cells.
Post-Thaw Colony Forming Unit (CFU) Counts and Yield Are the Most Important Predictors of Engraftment and Survival Following Unrelated Donor Cord Blood Transplantation (CBT): A COBLT Study Report.
Authors: Wall Donna A. , Wagner Elizabeth L. , Carter Shelly L. , Mendizabal Adam M. , Cairo Mitchell S. , Kernan Nancy A. , Laughlin Mary , Kurtzberg Joanne
Published: October 2019

Total nucleated cell count (TNC), CD34 and CFU content of cryopreserved cord blood units (CBUs) are measurements of hematopoietic potential previously demonstrated to be predictive of engraftment and/or survival post-transplant. Pre-cryopreservation measurements do not capture downstream events in cord blood banking that could impact CBT outcome: freezing, long-term storage, shipment to the transplant center and thawing. We hypothesized post-thaw enumeration of these measures would be more predictive of outcome. These tests were routinely performed at two of the COBLT transplant centers, at the time of CBU thaw. We examined the correlation of these laboratory measures pre-cryopreservation and post-thaw with engraftment and survival of 153 patients with leukemia and metabolic disorders undergoing myeloablative transplantation. The median age of recipients was 4.4 years (range 0.1 – 17.9) and weight 19.1 kg (range 3.9 – 118.4).

Pre-cryopreservation Thaw Yield Median (range) Median (range) Median (range) TNC/kg x 107/kg 6.9 (1.5 – 50.4) 5.7 (1.1 – 31.6) 80.9% (43.0 – 98.9) CD34/kg x 105/kg 2.3 (0.1 – 20.1) 2.0 (0.2 – 15.5) 72.6% (16.1 – 99.6) CFU/kg x 104/kg 20.2 (1.7 – 169.7) 3.0 (0.0 – 31.1) 17.7% (0.0 – 82.7) Trypan blue viability 99 (91 – 100) 98 (83 – 100) —

Overall 114/153 engrafted by day +42 with a median day to engraftment of 29 days. In multivariate analysis, post-thaw CFU count and yield were the highest correlates with engraftment (p<0.0001 and p<0.02, respectively) and survival (p=0.05 and p=0.02, respectively). If the CFU yield was 50–100% of pre-cryopreservation CFU then the probability of engraftment by day +42 was 93%. However, engraftment by day +42 fell to 63% when the yield was <5% (16% of CBUs). Survival at 2 years was poorer if CFU yield was less than 5% compared to >5% (38% vs. 65%, p =0.05). Importantly, post-thaw yield of TNC and trypan blue viability did not correlate with engraftment. Total hematopoietic content of units (TNC, CD34, and CFU) was strongly predictive of engraftment. However, independently, low CFU counts and poor yield of CFU at time of CBU thaw is a harbinger of poor/nonengraftment and is evidence of deterioration of the product between time of processing and infusion. Low CFU count or poor yield of CFU post-thaw may be used clinically to predict engraftment in patients undergoing CBT. These findings illustrate the need for the development of product release testing criteria for CBUs and should be a priority for the cord blood community.
Unrelated Bone Marrow Transplantation for Adult T Cell Leukemia/Lymphoma: A Study from the Japan Marrow Donor Program.
Authors: Kato Koji , Kanda Yoshinobu , Eto Tetsuya , Muta Tsuyoshi , Gondo Hisashi , Utsunomiya Atae , Taniguchi Shuichi , Shibuya Tsunefumi , Morishima Yasuo , Kato Shunichi , Kodera Yoshihisa , Harada Mine
Published: October 2019

Adult T cell leukemia/lymphoma(ATLL) is known to be a very poor prognosis, and intensified chemotherapy has not yet improved this prognosis. Some successful cases of allogeneic hematopoietic stem cell transplantation (allo-HSCT) from HLA-matched siblings have been reported and allo-HSCT has been suggested as improving the prognosis of ATLL. However, HLA-matched related donor is seldom available and about two-thirds of siblings of patients with ATLL are HTLV-1 carriers. Although most of ATLL patients require an unrelated donor to benefit from allo-HSCT, there is little information about the outcome of unrelated bone marrow transplantation(UBMT) for ATLL. To elucidate the feasibility and efficacy of UBMT from HTLV-1 seronegative donor, we retrospectively analyzed the data of 33 patients who were ATLL and underwent UBMT through the Japan Marrow Donor Program. Median age of patients at transplantation was 51 years(range,24–59). The conditioning regimens of 27 patients had myeloablative intensity and 6 had reduced intensity. At transplantation, 12 patients were in complete remission, 3 were partial remission and 14 were no remission. A total of 14 patients died after allo-HSCT, and 9 of those 14 patients were lost within 100 days after HSCT due to treatment related adverse events. The overall survival(OS), progression-free survival(PFS), cumulative incidences of disease progression, and nonprogression mortality at 2 years after UBMT were 49.7%, 42.6%, 24.5%, and 32.9%, respectively. Multivariate analysis demonstrated that recipient age(P=.044) and disease status at transplantation(P=.059) were independent prognostic factors for OS and PFS. These results suggest that UBMT could be considered as a treatment option for ATLL patients without an HLA-matched related donor. Further study is required to determine the indication of nonmyeloablative conditioning based on patient’s age or disease status, because myeloablative conditioning had high incidence of treatment related mortality.
Allogeneic Stem Cell Transplantation from Unrelated Donors within the Seventh Decade of Life.
Authors: Dahlke Joachim , Zabelina Tatjana , Ayuk Francis , Panse Jens , Schieder Heike , Renges Helmut , Atanackovic Djorde , Wolschke Christine , Zander Axel R. , Kroeger Nicolaus
Published: October 2019

We analyzed the outcome of 28 patients who were treated within prospective treatment protocols to investigate the feasibility of unrelated stem cell transplantation for patients with haematological malignancies within the seventh decade of life. Twenty-eight patients with a median age of 62 years (range 60–70) were enrolled. Twenty-six received a dose-reduced conditioning regimen while two patients were transplanted after standard conditioning regimen, and eight of the patients had received at least one prior high-dose chemotherapy followed by autologous or allogeneic stem cell transplantation. Diagnoses were leukaemia (AML: n=10; ALL: n=1; CML: n=2), MDS (n=5), myelofibrosis (n=3), multiple myeloma (n=6) or non-Hodgkin’s lymphoma (n=1). No primary graft-failure was observed, and the median number of days to leucocyte and platelet engraftment was 18 days and 23 days, respectively. Acute GvHD grade II–IV was seen in 35% of the patients, chronic GvHD was seen in 56% of the patients. The one-year cumulative incidence of treatment-related mortality was 25%. The four-year estimated overall- and disease-free survival was 49% and 40%, respectively. Unrelated stem cell transplantation in patients within the seventh decade of life is a feasible treatment option and may induce long-term disease-free survival.
Autologous Stem Cell Transplantation (ABMT) for Recurrent Hodgkin’s Disease from 1990–2005: Factors Contributing to Post-Transplant Relapse.
Authors: Bolwell Brian , Pohlman Brad , Kalaycio Matt , Andresen Steve , Kuczkowski Elizabeth , Brown Stacey , Bates Jennifer , Rybicki Lisa , Curtis Julie , Bell Kimberly , Sobecks Ronald M.
Published: October 2019

Long-term results of conventional therapy of Hodgkin’s disease (HD) has demonstrated the importance of long-term and ongoing follow-up given the potential for later complications after curative therapy. While many transplant series report follow-up of several years after ABMT, few report a 15-year experience from a single institution. This report examines the outcomes of 220 patients receiving high-dose chemotherapy and autologous stem cell transplant (ABMT) at The Cleveland Clinic Foundation from January 1990 through June 2005. Median age was 33 years (range, 14–70 years); median time from diagnosis to transplant was 19 months; 47% received prior radiation therapy; 82% had nodular sclerosis histologic subtype; number of courses of prior chemotherapy were: 1 (16%), 2 (66%), 3 (14%), 4 or more (4%). All patients received salvage therapy prior to transplant: 29% were in a complete remission (CR), 55% in a partial remission (PR), and 16% refractory. All patients received a chemotherapy-only preparative regimen, most commonly Bu/Cy/VP (73%), followed by CBV (17%). 78% received peripheral stem cells alone; 22% received either autologous bone marrow or a combination of bone marrow plus peripheral stem cells. At the present time 60% of patients are alive. Of the 88 patients who died, the most common cause of death is relapse (69% of deaths). Secondary malignancy occurred in 11 patients (5%); 9 of these cases were secondary AML/MDS and 5 of these patients with secondary malignancies have died. 44% of the entire cohort has relapsed, at a median of 9 months post-transplant (range, 1.4–76 months). 10-year overall survival is 47%. A multivariable analysis showed that the two significant variables that correlated with post-BMT relapse were the number of prior chemotherapies (p = 0.011), and patients treated in remission vs. those not in remission (p = 0.002). Of patients receiving 2 or more prior courses of chemotherapy, 60% have relapsed 8 years post-transplant, compared to 40% of those receiving one course of prior chemotherapy. The risk of relapse by the number of prior chemotherapy courses is shown graphically below:

Figure Figure

In conclusion, this very large series of ABMT for recurrent HD with long-term follow-up demonstrates the importance of timely autografting in relapsed HD patients. The optimal time to proceed with ABMT is after failing one, and only one, course of chemotherapy. Delaying transplant for unrealistic long-term salvage with other courses of traditional chemotherapy will negatively affect the outcome of subsequent ABMT.
Prognostic Value of FDG-PET in Lymphoma Patients Undergoing Autologous Stem Cell Transplantation.
Authors: Svoboda Jakub , Elstrom Rebecca , Chong Elise A. , Andreadis Charalambos , Berkowitz Arnold , Luger Selina M. , Porter David L. , Nasta Sunita , Downs Lisa H. , Siegel Donald L. , Glatstein Eli , Alavi Abass , Stadtmauer Edward A. , Schuster Stephen J.
Published: October 2019

High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard treatment for patients (pts) with relapsed or primary refractory lymphoma that is responding to salvage chemotherapy. Limitations of ASCT include significant morbidity, cost, restriction of further treatment options, and frequent relapse of lymphoma. To define the ability of FDG-PET to predict clinical outcome after ASCT, we conducted a retrospective analysis of lymphoma pts in our institution who received salvage chemotherapy followed by ASCT for relapsed or primary refractory lymphoma between 1999 and 2005. We identified 47 lymphoma pts (19 with diffuse large B-cell, 18 with Hodgkin, 4 with mantle cell, 3 with follicular and 3 with other lymphomas) who had an FDG-PET scan after at least two cycles of salvage chemotherapy and prior to ASCT. Three pts were excluded from the analysis because their FDG-PET scan results were non-conclusive. The remaining 44 pts (median age 44 yrs, range: 19–65) were categorized into FDG-PET scan negative (26 pts) and positive (18 pts) groups. Each group was evaluated for event-free survival (EFS), which was defined as the interval between the date of the transplant to the date of relapse after complete remission (CR) or the date of progression for pts without CR or the date of death from any cause without a documented relapse. The overall median follow-up was 14 mo (range: 2–58). There were no statistically significant differences in the distribution of lymphoma subtypes between the two groups. In the FDG-PET negative group, the median EFS was 18 mo (range: 3–58) with 14 pts (54%) remaining in CR. In the FDG-PET positive group, the median EFS was 5 mo (range: 1.5–19) with only 1 pt (6%) remaining in CR. Comparison between the two groups confirmed that the difference in the median EFS was highly statistically significant (p<0.001). Additional data to determine the correlation of the FDG-PET with conventional CT scans will be presented. Our study suggests that a positive FDG-PET scan after salvage chemotherapy and prior to ASCT indicates an extremely poor chance of achieving a prolonged remission after ASCT. Clinical trials of new approaches should be investigated for pts with relapsed or primary refractory lymphoma that remains detectable by FDG-PET after salvage chemotherapy.

Figure Figure
Allogeneic or Autologous Stem Cell Transplantation (SCT) for the Treatment of Pediatric Patients with Non-Hodgkins Lymphoma (NHL).
Authors: Ricafort Rosanna , Trippett Tanya , Kernan Nancy A. , Small Trudy N. , Prockop Susan , Scaradavou Andromachi , Keating Anna , O’Reilly Richard J. , Boulad Farid
Published: October 2019

Thirty six patients (pts) with NHL, aged 3.5–20.9 years (median 14.7 years) received an allo-SCT (n=21) or auto-SCT (n=15) at our institution between 12/82 and 12/04. Pathologic NHL classification included: lymphoblastic (n=12), Burkitt’s (n=5), Large Cell (n=17) including ki-1 + (n=11), peripheral NHL (n=1), and undifferentiated NHL (n=1). Disease status at SCT was: first remission (CR1) (n=1), CR2 (n=15), CR3 (n=5), partial remission (n=7), relapse (Rel) or refractory (Ref) (n=8). Cytoreductive regimens were total body irradiation (TBI)-based for all but 3 pts in each of the allo-SCT and auto-SCT group. For the allo-SCT pts, donors were: matched related (n=15), mismatched related (n=1), and unrelated (n=5); graft-versus-host disease (GvHD) prophylaxis included T-cell depletion (TCD) (n=10) or post-BMT immunosuppression (n=11). Only one pt received a non-myeloablative allo-SCT. With one pt in the allo-SCT group lost to follow-up, 35 of 36 pts in the entire cohort were evaluable. The median follow up of the entire group was 21.5 months; it was 34 months and 20 months respectively for the allo-SCT and the auto-SCT groups. The overall survival (OS) and disease-free survival (DFS) were 56% and 54%, respectively. Ten of the 11 pts with ki-1 positive NHL are alive, disease-free after either allo-SCT (n=4) or auto-SCT (n=7) with a median follow-up of 26 months. The DFS for those patients transplanted in CR was 60% compared to 49% for those transplanted in either PR or Rel/Ref disease. Of the eight pts transplanted with progressive disease, 2 are alive, disease-free at 110 and 115 months; both pts post allo-SCT. For the allo-SCT group, the DFS was 52%. Seven pts relapsed, 4 of whom had Rel/Ref disease at the time of SCT. Four pts died of SCT-related complications. Five pts developed Grade II–IV aGvHD. For the auto-SCT group, the DFS was 53%. Two pts relapsed, and 2 pts died of toxicity. In summary, both allo-SCT and auto-SCT offer the prospect of durable disease-free survival for a significant proportion of pediatric patients with NHL after a first relapse or disease progression. In particular, those pts with ki-1-positive NHL had the most favorable outcome. As expected, pts transplanted in minimal residual disease achieved superior DFS.

Results N CR/PR Rel/Ref Relapse TRM DFS Allo-SCT 21 11/4 6 33% 20% 52% Auto-SCT 15 10/3 2 13% 13% 53%
Measurement of ZAP-70 Expression in CLL Using an Optimized Flow Cytometric Assay for ZAP-70 Protein Levels in Whole Blood Samples.
Authors: Shankey T. Vincent , Cobb Jeffrey , Scibelli Paul , Smith Cecilia , Mills Rhonda , Forman Meryl
Published: October 2019

ZAP-70 protein plays a critical role in the initiation of T-cell signaling through the TCR, and also regulates B cell differentiation. Studies aimed at understanding the relationship of maturation to disease course in the B-cell form of Chronic Lymphocytic Leukemia (B-CLL) have demonstrated a correlation of disease progression with IgVH configuration (germ line vs mutated). Flow cytometry-based assays for ZAP-70 expression in CLL patient samples have been previously reported, and generally show poor separation of ZAP-70 levels in samples considered as positive from negative, making interpretation of these data difficult, and reducing the likely agreement between laboratories. Using a unique fixation and permeabilization technique in conjunction with an optimal ZAP-70-PE antibody conjugate, we have developed a whole blood assay for the measurement of ZAP-70 protein expression which results in a significantly increased S/N of 18–24 (normal blood T-cells to B-cells) compared to previously reported flow cytometric assays (S/N of 3 to 8). Based on these data, we have developed a 5 antibody/4 color whole blood assay (CD5-FITC, ZAP-70-PE, CD19-PE-Cy5, CD3+CD56-PECy7) which includes surface labeling before fixation and ZAP-70 labeling after permeabilization. Critical assay details including pre and post staining stability, instrument standardization, gating strategies and data interpretation will be presented and discussed. Using this optimized assay, we have undertaken a multi-institutional study to determine the inter-laboratory variability of ZAP-70 measurements using normal donors, and an assessment of the ability of this assay to increase the separation of B-CLL ZAP-70 positive from negative populations. Finally, we have developed a method to index differential ZAP-70 expression using internal controls, which obviates the use of quadrant analysis techniques.
Hemostatic Factors Regulate Immune Surveillance and Contribute to the Rapid Clearance of Bacterial Pathogens In Vivo.
Authors: Flick Matthew J. , Du Xinli , Talmage Kathryn E. , LaJeunesse Christine M. , Witte David P. , Palumbo Joseph S. , Degen Jay L.
Published: October 2019

The local activation of coagulation system proteases and secondary deposition of fibrin may constitute important “danger” or “damage” signals that promote leukocyte activation events at sites of infection or injury. Consistent with this general hypothesis, we previously showed that mice expressing a mutant form of fibrinogen lacking the leukocyte integrin receptor αMβ2-binding motif displayed compromised clearance of bacteria in a model of S. aureus-induced peritonitis. Here, we show that mice with a complete genetic deficiency in fibrinogen (Fib−/−) show an even more profound sensitivity to S. aureus peritonitis. Fib−/− mice were found to succumb to bacterial doses that were rarely fatal in wildtype mice following intraperitoneal injection of wildtype S. aureus. This profound difference in survival profile was also seen when mice were challenged with S. aureus lacking either the bacterial fibrinogen-receptor (ClfA) or the bacterial prothrombin activator, coagulase. These results indicate that efficient bacterial clearance in control animals is related to host leukocyte engagement of fibrin(ogen) as opposed to bacterial engagement of this hemostatic factor. Following intraperitoneal inoculation with one billion CFU of S. aureus, control mice successful cleared >99% of the bacteria within 1 hour, whereas the number of bacteria retrieved within peritoneal lavage fluid of Fib−/− mice remained similar to the original input CFU. Microscopy studies and bacterial fate studies revealed that the rapid loss of viable bacteria in control animals was not due to either fibrinogen-dependent dissemination to other tissues nor the simple association of live bacteria with peritoneal surfaces. Neutrophils and macrophages appear to participate in early bacterial clearance in animals of both genotypes, but these cells appear to be overwhelmed and engorged with microbes in Fib-/− mice. Leukocyte composition and trafficking into the peritoneal cavity was not appreciably altered in fibrin(ogen)-deficient mice. Furthermore, B cells, T cells, mast cells and complement factors C3 and C5 were not found to be major factors in the fibrinogen-dependent bacterial clearance. Taken together, the available data indicate that a failure to fully implement leukocyte-mediated antimicrobial mechanisms is the primary impediment to bacterial clearance in Fib−/− mice. The findings are consistent with the hypothesis that leukocyte engagement of provisional fibrin matrices within challenged tissues is a critical parameter in leukocyte “target recognition” in vivo.
Fibrinogen Hershey IV: A Novel Dysfibrinogen with a γ V411I Mutation in the Integrin αIibβ3 Binding Site.
Authors: Flood Veronica H. , Al-Mondhiry Hamid A. , Bakke Antony C. , Farrell David H.
Published: October 2019

The C-terminal segment of the fibrinogen γ chain plays a crucial role in platelet aggregation via its interaction with the platelet receptor αIIbβ3. It is well established that the last four amino acids of the γ chain (408 to 411) are critical for this function, as mutations or deletions of this region abrogate fibrinogen’s ability to bind αIIbβ3. We describe here the first naturally occurring fibrinogen mutation affecting the C-terminal region of the γ chain and investigate its effects on platelet interactions. The proband, a 49-year-old woman, was diagnosed with dysfibrinogenemia based on a prolonged thrombin time and low fibrinogen activity (55 mg/dL). Her bleeding history was significant for menorrhagia and one episode of post-operative hemorrhage. DNA sequencing of the fibrinogen genes demonstrated heterozygosity for two mutations, γ R275C and γ V411I. The latter γ V411I mutation represents a novel mutation affecting the C-terminal amino acid of the γ chain. We hypothesized that this mutation would decrease fibrinogen’s affinity for the platelet receptor αIIbβ3. In order to isolate the effects of this mutation on fibrinogen-platelet binding, γ 400-411 dodecapeptides were synthesized to mimic the C-terminal γ chain sequence. One peptide contained the wild-type sequence ending in valine (γ 400-V411), and the second peptide incorporated the isoleucine mutation (γ 400-I411). Previous studies have demonstrated that the wild type γ 400-V411 dodecapeptide inhibits platelet aggregation by competing for fibrinogen binding to αIIbβ3. We performed platelet aggregation studies comparing inhibition of aggregation with the wild-type γ 400-V411 and the mutant γ 400-I411 peptides. Washed platelets were obtained from a normal donor, and platelet aggregation monitored using the agonist ADP. The IC50 for the initial rate of aggregation with the γ 400-I411 peptide was 214 μM, compared to 133 μM with the wild-type peptide. We then examined the extent of aggregation in the presence of either wild-type or mutant peptide. Consistent with the previous results, total aggregation was lower with the wild-type peptide compared to the mutant peptide. The IC50 for the γ 400-I411 peptide was 450 μM compared to 250 μM with the γ 400-V411 peptide. Overall, these findings suggest that the γ I411 mutation results in a decreased ability to bind platelets. In the heterozygous state, however, the available amount of wild-type fibrinogen may be sufficient to support platelet aggregation. The bleeding diathesis observed for the proband could therefore reflect other factors, especially the γ R275C mutation.