bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced-Intensity Conditioning Allogeneic Hematopoietic Stem Cell Transplantation Represents a Feasible and Effective Treatment Strategy for Advanced Mycosis Fungoides and Sezary Syndrome.
Authors: Soligo Davide , Onida Francesco , Saporiti Giorgia , Volpe Aldo Della , Marino Cristian , Annaloro Claudio , Rambaldi Alessandro , Bruno Benedetto , Berti Emilio , Deliliers Giorgio Lambertenghi
Published: October 2019

Sezary syndrome (SS) and advanced tumor-stage mycosis fungoides (MF) have an aggressive clinical course, ultimately leading to a very short survival. Because patients are often elderly and show poor clinical conditions, conventional allogeneic hematopoietic stem cell transplantation is associated with a undesirable high TRM; therefore, allogeneic reduced-intensity conditioning (RIC) HSCT represent an attractive and worth-investigating strategy of cure in this disease setting. In our Institutions, from September 2000 to May 2006, fifteen patients with advanced mycosis fungoides or Sezary Sindrome underwent RIC allogeneic hematopoietic stem cell transplantation either from HLA-identical sibling, or from HLA-matched unrelated donor, or from unrelated cord blood. Conditioning regimens for patients with HLA-identical sibling included fludarabine/cyclophosphamide/TBI200 up to 2001, then pentostatin/TBI200. Patients who underwent unrelated transplants were treated with melphalan/CP1H/Fludarabine/TBI200 combination. All patients (10 males and 5 females) had a clinical diagnosis of MF (10 pts) or SS (5 pts) confirmed by histopathology, immunohistochemistry and molecular biology. Median age was 48 years (range 38–65). Median time from diagnosis to transplant was 48 months (range 13 to 252). Disease stage for all patients was from IIIB to IVB. GVHD prophylaxis included oral Cy-A from day 0 to day +100 and oral MMF from day 0 to day +27, then tapered within 2 weeks. The source of donor stem cells was peripheral blood and bone marrow for siblings and MUD, respectively. A complete clinical remission was achieved in 10 patients, whereas 2 patients obtained a VGPR. Five patients died from various complications (3 with progressive disease, 1 in CR e 1 in PR) within a period ranging from 33 days to 8 months after transplant. A full donor chimerism was achieved within 6 months in 8 out of 10 evaluable patients, whereas no engraftemnt was observed in 2 patients (1 MUD and 1 UCB). 5 patients experienced acute GVHD of grade I–II, while limited chronic GVHD occurred in 7 patients. With a median follow-up of 38 months (range 2–70), ten patients were alive with an estimated overall survival greater than 60% at five years (Figure 1), whereas 7 patients were alive and disease-free at the last follow-up. Based on these highly encouraging results, we conclude that RIC allogeneic HSCT represents a feasible and effective treatment for advanced mycosis fungoides and Sezary Syndrome. Further studies in larger patient populations are warranted to confirm the curative potential of this strategy.

Overall Survival in MF/SS following RIC allo-HSCT Overall Survival in MF/SS following RIC allo-HSCT
Tissue Plasminogen Activator (t-PA) and Plasminogen Activator Inhibitor-1 (PAI-1) in Essential Thrombocythemia and Polycythemia Vera and Thrombotic Complications.
Authors: Cacciola Rossella R. , Di Francesco Ernesto , Giustolisi Rosario , Cacciola Emma
Published: October 2019

Thrombotic complications are frequent in patients with essential thrombocythemia (ET) and polycythemia vera (PV). While a thrombin- and tissue plasminogen activator (t-PA)-regulated release of t-PA and plasminogen activator inhibitor-1 (PAI-1), respectively, has been shown and an association between elevated t-PA and PAI-1 and thrombogenesis has been studied for several vascular diseases, few studies have concentrated on the correlation between t-PA and PAI-1 levels and thrombogenesis in these myeloproliferative disorders. Therefore, we investigated prothrombin fragment 1+2 (F1+2), as marker of thrombin generation, and t-PA and PAI-1 in patients with ET and PV and thrombosis. We included 44 patients, 22 ET (5 men and 17 women, mean age 55 years) and 22 PV (17 men and 5 women, mean age 60 years) who fulfilled PVSG. The mean duration of disease was 4.5 years. Most received cytoreduction, such as hydroxyurea (13 ET and 14 PV), interferon-alpha (2 ET), anagrelide hydrochloride (5 ET) or phlebotomy (8 PV). None of studied patients had thrombotic risk factors. Of 44 patients, 25 (12 ET and 13 PV) developed thrombosis, whereas 19 (10 ET and 9 PV) did not. Measurements were assayed by ELISA. All patients had increased F1+2 (2.8±2.8 nmol/L vs 0.7±0.2 nmol/L) (p=0.001). t-PA and PAI-1 were higher in those patients with previous thrombosis than in asymptomatic patients (130±62 ng/ml vs 89±69 ng/ml and 45±20 ng/ml vs 57±27 ng/ml, respectively) (p=0.043 and p=0.085, respectively). A positive correlation there was between F1+2 and t-PA and t-PA and PAI-1 (p<0.0001 and p<0.0001, respectively). These findings suggest that a disfibrinolysis may be in ET and PV and that t-PA and PAI-1 may be predictive additional factors of the thrombotic tendency of these patients.
Experimental Study on Prevention of GVHD by Selective Elimination of Alloreactive Donor Lymphocytes Prior to Stem Cell Transplantation.
Authors: Chen Bao-An , Dong Wei-Min , Ding Jia-Hua , Deng Xiao-Jing , Zhao Gang , Gao Chong , Sun Yun-Yu , Wang Jun , Cheng Jian , Chen Ning-Na , Fei Fei
Published: October 2019

Objective: To investigate a new concept aiming for induction of graft-vs-leukemia (GVL) effect prior to stem cell transplantation (SCT). Mismatched lymphocytes given pre-SCT will be followed by selective elimination of alloreactive donor lymphocytes, thus avoiding lethal graft-vs-host disease (GVHD).

Methods: Female (BALB/c×C57BL/6)F1 mice (H-2d/b) as recipients received sublethal total body irradiation (TBI) of 4 Gy (60Coγ-ray) on day 0 followed by being inoculated with 0.5×107 P388D1 leukemia cell line on day 1, injection of 1.5×107 allogeneic splenocytes supplied by C57BL/6 male mice(H-2b)for induction of GVHD, intraperitoneally injection of cyclophosphamide (Cy) (200 mg/kg) or TBI (9 Gy) were given on day 7, one day later, treated mice were rescued with 3×107 syngeneic bone marrow cells supplied by (BALB/c×C57BL/6)F1 male mice(H-2d/b). Recipients were observed clinical manifestation, phenotype, re-establishment of haematogenesis, histopathologic changes of internal organs suffered from GVHD and investigated donor chimerism by the semi-quantitate analyses of polymerase chain reaction (PCR). Data was analyzed by SPSS 10.0 software and expressed as mean ± SD.

Results: Recipients had no occurrence of leukemia and GVHD by selective elimination of alloreactive donor lymphocytes by Cy and TBI, survived more than 210 days, to become complete-donor chimerism on day +21. The ratio of chimerism descended subsequently, but still displayed mixed-chimerism on day +90. Control mice died of evident GVHD, leukemia or other death-related-transplantation within 20 to 36 days(p<0.01). Attempting to induce GVL effects by mismatched lymphocytes given before stem cell transplantation followed by selective elimination of alloreactive donor lymphocytes, thus avoiding graft-vs-host disease (GVHD) was feasible.
The Role of Selective Depletion of Alloreactive Donor T Cells in Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation.
Authors: Chai Yihuan , Qiu Huiying , Lv Hui
Published: October 2019

One of the main goals in allogeneic bone marrow(BM) transplantation is the abrogation of graft-versus-host disease (GVHD) with the preservation of antileukemia and antiviral activity. The Study present a selective T cell depletion strategy based on the physical separation of the alloreactive T cells, which were identified by expression of two activation-induced antigens (CD25 and CD69). T cells from C57BL/6(H-2b) mice were first activated with BALB/c (H-2d) recipient spleen cells in a 2-day mixed-lymphocyte-culture (MLC). Following this activation, this compound is selectively depleted based on expression of two activation-induced antigens CD25 and CD69 using magnetic cell sorting. The depleted cells or the untreated cells were then rechallenged respectively in a secondary MLC, with the same stimulator cells or a third-party (DBAH-2k) or tumor- specific (SP2/0, BALB/c-origin myeloma) cells. Cells proliferation were assayed at the indicated time points(1, 2, 3, 4, 5 days). These treated cells or control-cultured cells (2.0×106) mixed with 5.0×106 BM cells from C57BL/6 were transfused respectively by the trail vain into the lethally irradiated BALB/c to observe the survival time, GVHD incidence and pathological analysis. MLC assays demonstrated that this technique led to a significant decrease in alloreactivity of donor cells(29.02~64.17%), which at the same time preserved reactivity against third party cells(49.61~75.69%)and anti-tumor cells(61.14~68.62%). The mice in the group of control-coclutured were died of acute GVHD within 24days. The 7 recipient mice in the treated group were free of acute GVHD, and 3 mice were died of acute GVHD (aGVHD) within 23 days. MACS-based ex-vivo depletion of alloreactive donor T cells based on expression of two activation-induced antigens (CD25 and CD69) could inhibit anti-host responses, by contrast, anti-SP2/O and anti-third-party responses were preserved. Cotransplantation of these selected depleted cells and BM cells could reduce aGVHD.
Double-Stranded RNA Acts as a Strong Danger Signal in Human Myeloid Leukemia Cells Leading to Increased Immunogenicity.
Authors: Berneman Zwi N. , Smits Evelien L.J.M. , Ponsaerts Peter , Cools Nathalie , Van de Velde Ann L.R. , Gadisseur Alain P. , Schroyens Wilfried A. , Van Tendeloo Viggo F.I.
Published: October 2019

Leukemic cells exert immunosuppressive effects that interfere with dendritic cell function and hamper effective anti-leukemic immune responses. Recently, Toll-like receptor 3 (TLR3) was characterized in dendritic cells as an intracellular double-stranded (ds)RNA receptor which is triggered by viral infection or incubation with the synthetic dsRNA analogue polyriboinosinic polyribocytidylic acid [poly(I:C)], leading to maturation and activation of dendritic cells. Until now, little was known on the expression of TLR3 in leukemic cells and their responsiveness to dsRNA treatment. We assessed TLR3 expression in primary and transformed acute myeloid leukemia (AML) cells and hypothesized that the immunogenicity of AML cells could be improved by treatment with the synthetic TLR3 agonist poly(I:C), thereby mimicking viral infection of these leukemic cells. In view of this hypothesis, we electroporated or pulsed transformed and primary AML cells with poly(I:C) and analyzed the effect of poly(I:C) loading on TLR3 expression, costimulatory molecules, cytokine production and allogeneic T cell response. We also assessed the uptake of poly(I:C)-loaded leukemic cells by immature dendritic cells and the subsequent effect on dendritic cell activation and maturation status. We observed that primary and transformed AML cells respond to poly(I:C) electroporation by upregulation of TLR3 expression, apoptosis, elevated levels of costimulatory molecules CD80 and CD86 and by production of type I interferons (IFN). Furthermore, poly(I:C)-electroporated AML cells induced interferon-gamma production by allogeneic T cells. Upon phagocytosis of poly(I:C)-electroporated AML cells, dendritic cells showed an increased expression of maturation markers and marked production of proinflammatory cytokines. In contrast, this set of immune effects was absent or suboptimal when AML cells were passively pulsed with poly(I:C), indicating the superiority of transfection over pulsing with poly(I:C). These results demonstrate that poly(I:C) electroporation is a promising novel strategy to increase the immunogenicity of AML cells.
Partly MHC Matched Allogeneic Tumor Specific T Cells Mediate Tumor Regression without Inducing GVHD in Immunosuppressed Host.
Authors: Boni Andrea , Muranski Pawel , Wrzesinski Claudia , Kaiser Andrew , Paulos Chrystal , Palmer Douglas , Gattinoni Luca , Restifo Nicholas P.
Published: October 2019

Adoptive cell transfer (ACT) immunotherapy following lymphodepleting conditioning is a promising strategy for the treatment of metastatic solid tumors, however the difficulties in generating autologous tumor specific lymphocytes for every patient has significantly limited its applications. Allogeneic partly matched tumor specific T cells could be used for patients in whom autologous cells are not available, however their rapid rejection by the host restricts this approach. When CD8+ pmel-1 T cells from B6-BALB-C F1 (b/d) were transferred into B16 tumor-bearing B6 mice (b/b) or into a different F1 (b/k), these tumor-specific T cells were rapidly rejected, and had no impact on the tumor regression. Here we show that following myeloablative conditioning, the adoptive transfer of allogeneic, major histocompatibility mismatched tumor-specific T lymphocytes resulted in the regression of large vascularized tumors. The ability of adoptively transferred allogeneic T cells to mediate tumor regression was directly proportional to the dose of irradiation given prior T cell transfer which also correlated with the in vivo survival of the transferred cells. At the highest irradiation dose used (i.e. 11 Gy) allogeneic T cells could survive for as long as 4 weeks and their efficacy was comparable to syngeneic tumor-reactive T cells. In addition we found that the risk of inducing a graft versus host (GVH) reaction was minor when the specificity of transferred TCR is confined. In fact co-transfer of transgenic cells and small amounts of open repertoire T cells (2*10e4) able to react with the host did not result in any measurable toxicity whereas co transfer of greater quantities (2*10e5 or more) could cause fatal GVHD effect. Interestingly GVH reaction was associated with an improved tumor treatment, though this effect was transient as most of the animals succumbed to GVHD. Here we demonstrate that allogeneic T cells might represent an important tool in cancer immunotherapy allowing treatment of patients for whom it is not possible to obtain autologous cells. Furthermore the possible synergy between tumor specific allogeneic T cells and GVH effect could be exploited in bone marrow transplant protocols.
AMD3100 Plus G-CSF Mobilizes the Majority of Non-Hodgkin’s Lymphoma (NHL), Multiple Myeloma (MM), and Hodgkin’s Disease (HD) Patients Who Failed Prior Mobilization with Other Regimens.
Authors: Calandra Gary , McCarty John , McGuirk Joseph , Barlogie Bart , Crocker Sue-Anne , Badel Karin
Published: October 2019

Background: AMD3100, an inhibitor of SDF1 binding to CXCR4, synergizes with G-CSF to allow mobilization of sufficient CD34+ cells/kg for autologous transplantation in pts unable to collect adequate CD34+ cells with G-CSF alone. Therefore, a single patient use (SPU) protocol for AMD3100 was adopted for more than one patient entry per site and termed Compassionate Use Protocol (CUP). The only difference of standard of care was the addition of AMD3100 to a G-CSF mobilization on the evening prior to each day of apheresis. Pts who could not proceed to apheresis due to low peripheral blood counts or pts who did not collect a minimum of 2 × 10^6 CD34+ cell/kg were eligible.

Methods: Overall, more than 280 patients with proven poor mobilization including 137 NHL, 73 MM, and 31 HD have been included in CUP. A data audit was performed to validate data from pts with either non-Hodgkin’s lymphoma (NHL), multiple myeloma (MM), or Hodgkin’s Disease (HD). Sites were selected based on most patients entered, >3 of all diseases entered at a site, and sites conducting an AMD3100 trial. Audit included all pts, regardless of success or failure of the outcome; all information was collected on CRFs. Success of outcome was collection of ≥ 2×10^6 CD34+ cells/kg during the CUP procedure. CUP apheresis was done on day 5 after G-CSF (10mg/kg SC QD) and AMD3100 (240mg/kg SC Q10 PM) starting day 4.

Results: Charts and CRFs were available for review for 115 pts; 63 (55%) were NHL patients from 29 sites, 35 (30%) were MM from 23 sites and 17 (15%) were HD from 13 sites. Of these pts, 58% were male, the median age was 59 years (range 21–77), 88% were Caucasian, and patient weight ranged from 43–128 kg. Prior treatments included a median of two regimens of chemotherapy in each of the three groups. Safety was generally favorable with no drug-related SAE’s and with an AE profile similar to that seen in research trials. The rates of successful collection of ≥ 2×10^6 CD34+ cells/kg per disease state as well as prior mobilization regimen are summarized in table 1. The median number of mobilizations for the successful patients was 3 days for NHL and HD and 4 for MM. Eighty-eight of the patients underwent transplantation with any cells. For example, of the 47 NHL patients transplanted, 24 had CUP only cells and 23 had mixed cells. The median number of days to engraftment was 11 for PMN and 18 for platelets. Long term follow up is limited, but there do not appear to be graft failures. At least 12 of the pts have died.

Conclusions: AMD3100 in a poor mobilizer population is generally safe and well tolerated and is very effective in mobilizing > 2×10^6 CD34+ cells/kg.

Mobilization success rate by disease state: overall and prior mobilization regimen Overall Prior Cytokine Prior Chemotherapy NHL 60% 53% 68% HD 76% 78% 75% MM 71% 73% 71%
Donor T-Cell Chimerism Does Not Predict Relapse and Response to Donor Lymphocyte Infusion for a Patient of Secondary Acute Myeloid Leukemia after Reduced-Intensity Allogeneic Hematopoitic Stem Cell Transplantation.
Authors: Tang Xiaowen , Zhang Qiurong , Wu De Pei , Feng Yufeng , Sun Aining
Published: October 2019

We analyzed donor chimerism(DC) in unfractionated(UF) and CD3+ cells from a secondary acute myeloid leukemia patient who relapsed after reduced-intensity allogeneic hematopoitic stem cell transplantation(RIST) and had undergone donor lymphocyte infusion(DLI). Sequential and quantitative chimerism was performed by multiplex PCR amplification of STR markers (STR-PCR). The peripheral blood(PB) every 3 days and bone marrow every week were collected for chimerism analysis. The patient presented hematological relapse 26 months after the first RIST. There were 31.5% blast cell in the bone marrow smear and presented additional abnormal chromosome [44,XY,-7,dic(16,17)]. While UF donor chimerism from PB and CD3+ fraction donor chimerism from PB and BM were always full donor chimerism (range, 96.4%~98.1%). However UF chimerism from BM was 64.9% at the same time point. Afterwards the patient received 4 times of dose-escalated donor lymphocytes infusion (DLI) with CD3+ cell from 1*107/kg to 1.0*108/kg. Unfortunately the patient hasn’t response to this therapy. The blast cell in BM smear augment from 31.5% to 51.5%. UF chimerism from BM ranged from 55.2 to 46.5%. But UF DC from PB and CD3+ fraction DC from PB and BM ranged from 95.5% to 98%. In conclusion, Our result suggest that in this AML patient the donor T-cell chimerism and unfractionated chimerism in PB can not predict relapse and response to donor lymphocyte infusion. and the reason is not clear.
Transient Mixed Chimerism after Stem Cell Transplantation Can Induce Durable Molecular Complete Remissions in Chronic Myelogenous Leukemia.
Authors: Ruiz-Arguelles Guillermo J. , Gomez-Almaguer David , Ruiz-Delgado Guillermo J. , del Carmen Tarin-Arzaga Luz
Published: October 2019

Three patients with Ph1 (+), BCR/ABL+ chronic myelogenous leukemia were allografted, two with unrelated compatible placental blood cells and one from an HLA compatible sibling. The three patients engrafted successfully, achieved mixed chimerism and all cleared the BCR/ABL fusion transcript. Despíte the fact that the three patients lost the partial chimerism, they have remained in complete molecular remissions 7 months, fourteen months and five years after the allografts. It is possible that the iatrogenic induction of a transient chimerism and in turn of a transient graft versus leukemia effect, might have a role in the induction of the sustained molecular remission of these three individuals with CML despite the fact that the three patients lost the graft; however other possible explanations can be offered. A longer follow up of the patients is mandatory to further clarify these observations.
Disappointing Outcomes of Peripheral T Cell Lymphoma after Autologous Stem Cell Transplantation.
Authors: Smith Stephen D. , Sweetenham John William , Rybicki Lisa , Brown Stacey , Dean Robert M. , Kalaycio Matt , Sobecks Ronald , Andresen Steve , His Eric , Pohlman Brad , Bolwell Brian J.
Published: October 2019

The role of high dose therapy and autologous stem cell transplantation (ASCT) for patients with peripheral T-cell lymphoma (PTCL) is poorly defined. Comparisons of outcomes between PTCL and B-cell NHL following HDT have yielded conflicting results, in part due to the rarity and heterogeneity of PTCL. Older retrospective studies found comparable survival rates after ASCT for pts with T-cell and B-cell NHL.1,2 In this study, we report our single center experience over one decade using a uniform high-dose regimen for patients with PTCL.

Patients and Methods

The transplant database of the BMT program at Cleveland Clinic was reviewed, and 32 patients undergoing ASCT for PTCL between 1996 and 2005 were identified. Twenty-one patients (66%) had anaplastic large cell lymphoma (ALCL), and 11 (34%) had peripheral T cell, not otherwise specified (PTCL-NOS). Patient characteristics are summarized in table 1.

Stem cell mobilization with VP16 and GCSF priming provided a median CD34 cell dose of 5.01× 106/kg (range 2.05–29.69). Patients received a preparative regimen consisting of busulfan (either 1 mg/kg orally or 0.8mg/kg IV for 14 doses), followed by VP16 60 mg/kg IV continuous infusion, then cyclophosphamide 60mg/m2 IV daily for two days. Standard supportive care measures were employed.

Results

Recovery to 500 neutrophils/uL occurred at a median of 10 days post transplant (range 9–12 days) and platelet recovery to 20 000 at a median of 14 (range 7–60) days.

Kaplan-Meier 5 year overall survival and relapse-free survival for all patients is 34% and 18%, respectively; median survival for all patients is 36 months (see figure 1). Median follow-up of 10 survivors is 25 months. No obvious plateau was observed on the overall or relapse fee survival curves. No significant difference in outcomes based on subgroup (ALCL versus PTCL-NOS) was observed. Staining for anaplastic lymphoma kinase (ALK) was available for 11 (of 21 total) anaplastic T cell lymphoma patients: 4/5 ALK-positive patients are alive compared to 2/6 ALK-negative patients at last follow-up. Four of five patients undergoing ASCT as consolidation following initial therapy are alive at a median 25 months.

Based on this small patient population, and in contrast to some recent studies, our results suggest a poor outcome for patients with PTCL after ASCT. The outcome for pts undergoing ASCT in first remission, and for ALK-positive (versus ALK-negative) ALCL, requires prospective investigation.

Table 1: Patient Characteristics Characteristic N (%) ALCL 21 (66) PTCL NOS 11 (34) Male 21 (66) Age: median(range) 44 (16-69) 2 prior chemo regimens 22 (69) 3 or more prior chemo regimens 7 (22) Transplant in first remission 5 (16) Relapsed/Refractory 25 (78)

Figure Figure
Determination of the Maximum Tolerated Dose of Cyclophosphamide in Conjunction with High-Dose Etoposide and Carboplatin Followed by Autologous CD34+ Hematopoietic Rescue in Pediatric Patients with Recurrent/Refractory Solid Tumors.
Authors: Hudspeth Michelle P. , Duffy Barbara , Noga Stephen , Goodman Steven , Civin Curt I. , Chen Allen R.
Published: October 2019

Cyclophosphamide (Cy) is an alkylating agent utilized in almost all preparative regimens for hematopoietic stem cell transplantation, and in vitro sensitivity has been demonstrated with carboplatin and etoposide. Multiple previous studies have involved this combination of agents; however, they almost exclusively utilized a fixed dose of Cy and/or bolus administration. The potential fatal cardiac toxicity of Cy is likely related to high peak concentrations of active metabolites from bolus dosing. Additionally, some evidence exists that the increased metabolism of Cy with continuous infusion has a therapeutic benefit. The objective of this study was to determine the maximum tolerated dose (MTD) of continuous infusion Cy with fixed dose etoposide (2400 mg/m2) and carboplatin (2175 mg/m2) followed by autologous CD34+ hematopoietic rescue in pediatric patients with recurrent/refractory solid tumors. Twenty patients were enrolled, and fourteen patients completed therapy on study. The MTD of Cy was 60 mg/kg/24 hr by continuous 60 hr infusion on days -4 to -2 for a total dose of 150 mg/kg. Alopecia, stomatitis and nausea were the most common toxicities. The single episode of direct cardiac toxicity was reversible. Two Grade IV toxicities occurred, consisting of diarrhea in one patient and coma in another patient who recovered and remains a long-term survivor. All patients were evaluable for neutrophil engraftment, with a median time to ANC > 200/μl of 11 days (range 7–30 days). Thirteen of the fourteen patients were evaluable for platelet engraftment, with a median time to platelet count of 20,000/μl independent of transfusion of 21 days (range 11–72 days). Six out of the fourteen patients remain alive greater than five years after study completion. The MTD of Cy was lower than expected. These results suggest that autoinduction of Cy metabolism (repeated administration of Cy at 24 hour intervals leads to increased hepatic cytochrome enzymes) could be a primary factor in the determination of the MTD. Future studies should aim to further characterize the pharmacokinetics and effectiveness of continuous infusion Cy in pediatric patients.
Dose Dense Dreifach* Melphalan100 for Multiple Myeloma.
Authors: Berz David , McCormack Elise M. , Winer Eric S. , Karwan Patricia , Colvin Gerald , Rathore Ritesh , Lum Larry , Abedi Mehrdad , Elfenbein Gerald , Quesenberry Peter J.
Published: October 2019

Background: Tandem high dose melphalan therapy with autologous peripheral stem cell support has emerged as standard of care for patients without prohibitive comorbidities. Mucositis and gastrointestinal side effects are the most common extrahematologic side effects. Two previously published studies presented a triple transplant with a conditioning regimen melphalan 100mg/m2 (MEL100) with peripheral stem cell support every two to five months schedule. This dose reduced regimen was implemented to limit those extrahematologic dose limiting side effects in the elderly or high risk population. We are presenting a novel approach that investigates the triple melphalan100/m2 approach on a dose dense, every three weeks schedule in a patient population without significant comorbidities for conventional high dose tandem transplantation.

Patients and methods: Thirteen standard or high risk patients with stageIII multiple myeloma were prospectively treated This population contained eight patients with IgG clonality, 3 IgA, 1 nonsecretory and one light chain isotype. The induction regimens of the thirteen patients were heterogenous and included 5 VAD, 3 DCIE, 2 Thal/Dex, 2 CIE and 1 pulse decadron. Patients had stem cells harvested through peripheral blood leukopheresis, and these cells were divided into three equal sets and frozen. The patients were scheduled to receive Melphalan at 100mg/m2 on day -1, 20, and 41 and then the autologous infusions occurred at day 0, 21, and 42.

Results: All patients were able to receive all three cycles of the MEL100 regimen. Seven patients (54%) received the treatements on the every three week schedule; three treatments (23%) during the second cycle and six treatments (46%) of the third cycle had to be delayed a median of 6 and 4 days respectively. Three patients were managed completely in the outpatient setting, and the average total hospital stay for the three transplants was 18 days. Median progression free survival was 761 days (range 73 – 1571) and the overall survival of this cohort has yet to be reached. No patient had worse than grade II mucositis, and no serious adverse events were recorded.

Conclusions: Our regimen of three consecutive autologous peripheral stem cell transplants with a reduced dose of melphalan at 100mg/m2 given every three weeks is very well tolerated. The progression free survival and overall survival are similar and can be compared favorably with the standard tandem myeloma regimens. Although our data is intriguing, further studies with larger numbers need to be performed to confirm these results.
Novel Gene Delivery System of Sendai Virus Transduced Human Peripheral Monocytes.
Authors: Tanaka Tomoko , Kurita Ryo , Takasugi Kashiya , Nakamura Takafumi , Inoue Makoto , Hasegawa Mamoru , Ono Fumiko , Terao Keiji , Yoshikawa Yasuhiro , Tani Kenzaburo
Published: October 2019

Extensive monocytes extravasation is seen in infection sites, inflamed tissue and tumor tissues and exhibit a tissue-specific range of functions including phagocytosis, antigen presentation to T cells, and the release of a wide array of cytokines, chemokines, enzymes and nitrogen species. As monocytes have natural tropism to such tissues in each disease condition, these cells can be used as novel gene delivery system to cure the condition. On the other hand, as monocytes are quiescent cells, available gene transfer vectors are limited. Sendai virus (SeV) is a negative-strand RNA virus and has recently been used for the gene therapy vectors, SeV vectors (SeVV), for somatic gene therapy. This vector does not have potential of stable transformation of target cells because they generate no DNA intermediates and therefore are unable to integrate. Also, SeVV has been shown to ensure efficient expression of foreign genes in various types of tissues and can be used for gene transfer into monocytes. We first of all studied the gene transduction efficiency of SeVV into human peripheral blood CD 14 positive monocytes using GFP gene as a marker. About 82% of monocytes became GFP positive, whereas only 23% of lymphocytes and 1.8% of granulocytes became positive. The transduction efficiency was peaked at M.O.I.=1. The expression level was at least preserved for 24 hours in vitro. To do the in vivo preclinical experiments, we also transduced cDNA of GFP and GDNF (glial cell line-derived neurotrophic factor) into the peripheral monocytes of Macaca fascicularis monkey and high level expression of these genes were obtained. In vitro monocyte function after gene transduction are now under investigation. SeVV is considered to be promising gene transfer vector for primate monocytes.
Impact of Hydroxyurea on Thrombin Antithrombin Complex and Vaso-Occlusive Crisis in Pediatric Sickle Cell Disease.
Authors: Elalfy Mohsen Saleh , Abdelmonem Ashraf M. , Youssef Soha , Ismail Heba
Published: October 2019

Background: Several studies suggest that increased activity of the coagulation system may be important in the pathogenesis of vascular occlusion in sickle cell disease. Hydroxyurea (HU) has been shown to reduce the frequency of vaso-occlusive manifestations in both adults and children with sickle cell disease (SCD).

Aim: To analyze the effect of HU on Thrombin-Antithrombin (TAT) as a marker of thrombin generation and hypercoagualbility in SCD and to find out the relation between TAT level and vaso-occusive crisis.

Subjects and Method: we evaluated 37 child with sickle cell hemoglobinopathy (mean age 10.92±5.39 years) and 15 normal control children (mean age 9.75±6.34 years). Informed consent was obtained from patients and/or guardians and study approval by local IRB was obtained. Twenty-two patients (59.5%) were on HU, 15 (41.5%) patients did not receive HU, 7 (46.7%) of them were transfusion dependant. TAT assay was done in vitro using a sandwich enzyme immunoassay.

Results: Mean patients’ age at institution of HU was 8.54± 3.85 years with median treatment duration of 4.5 years. Causes for initiating HU therapy were frequent blood transfusion in 11 patients (50%), frequent pain crisis (≥ 3/year) in 9 patients (41%), severe anemia and parents refusing blood transfusion in 1 patient (4.5%) and stroke in another patient (4.5%). HU dose was 20.82±4.95 mg/kg/day. We measured TAT in all patients and compared them to healthy control. There was significant difference in TAT level in sickle cell patients (198.86±185.7) compared to healthy control 2.91±0.94, [P value < 0.0001]. When the level of TAT was compared between the HU and non-HU groups we found that patients on HU had statistically significant lower TAT level (172.36 vs.225.37) [P=0.039]. There was also a significant negative correlation between HU dose and TAT level (p=0.03). A significant positive correlation between number of vaso-occlusive crisis/year [P=0.03], frequency of pain crisis/year [P=0.04], duration of pain crisis [P=0.03] and TAT level was observed.

Conclusion: Hydroxyurea has significant inhibitory effect on thrombogenesis in sickle cell patients, which may be another mechanism for reducing vaso-occlusive crisis. Sickle cell children with higher TAT level had more frequent and severe vaso-occlusive crisis.
Impact of Clinical/Demographic Characteristics and Duration of Clinical Benefit on Average Weekly Doses of Darbepoetin Alfa and Epoetin Alfa in Oncology: Results from a Claims Analysis.
Authors: Song Xue , Long Stacey R. , Kallich Joel D. , Marder William D.
Published: October 2019

Background: Comparison of darbepoetin alfa (DA) and epoetin alfa (EA) has often relied on calculation of unadjusted average weekly doses (AWD). This study examined the impact of addressing differences in clinical/demographic characteristics and duration of clinical benefit (DCB) of last dose administered on AWD.

Methods: MarketScan claims data were used to build EA and DA treatment episodes among cancer patients with >= 1 EA or DA claim and continuously enrolled January–June 2005. AWDs were calculated based on 2 different DCB scenarios (DCB1 and DCB2) for the last dose administered in the episode. DCB1 assumed DA doses <=100mcg =7 days, 101–299mcg =14 days, and >=300mcg =21 days; and EA doses <35,000units =2 days and >=35,000units =7 days. DCB2 assumed DA doses <60mcg =2 days, 60–149mcg =7 days, 150–299mcg =14 days, and >=300mcg =21 days; EA doses <=15,000units =2 days, 15,001–35,000units =5 days, and >35,000units =7 days. Logistic regression examined if clinical/demographic characteristics (age, gender, cancer type, comorbidities, and metastases) influenced likelihood of DA vs EA use; ANOVA models estimated an adjusted AWD controlling for these clinical/demographic differences.

Results: 2,942 DA and 2,574 EA episodes were created. Younger patients, with more comorbidities, and advanced cancer were found to have increased likelihood of DA use (all p<0.001). The DCB analysis revealed that EA was more sensitive to DCB changes than DA. Switching from DCB1 to DCB2 increased AWD for DA from 94mcg to 105mcg, while EA AWD decreased from 44,044units to 35,496units. With ANOVA adjustment to equalize the populations, estimated AWD for DCB1 and DCB2, respectively, were 97mcg and 104mcg for DA and 41,902units and 34,973units for EA.

Conclusions: Equalizing clinical/demographic characteristics and addressing possible DCB differences for DA and EA can substantially change the average weekly doses calculated from claims data; unadjusted AWDs may be misleading. In addition, comparisons of dose using claims data must be interpreted with caution because such data lack information on patient clinical outcomes of erythropoiesis stimulating agents and are subject to bias including data recording errors and confounding by indication.
Efficacy, Safety and Tolerability of a Novel High-Purity, Double Virus-Inactivated VWF/FVIII-Concentrate in the Treatment of Von Willebrand Disease (VWD) - Clinical Experience in the Treatment of Bleeding and Surgery.
Authors: Schwartz B.A. , Windyga J. , von Depka M. , Walter O. , Jansen M. , Auerswald G.
Published: October 2019

Introduction: Wilate is a highly purified, double viral inactivated, VWF/FVIII-concentrate developed for the treatment of VWD. A program of prospective studies was conducted in VWD patients to investigate the clinical efficacy and safety of Wilate® in acute bleeding episodes, prophylaxis and surgical procedures.

Methods: In these prospective open label studies 70 VWD patients (37 VWD of type 3) were treated with the VWF/FVIII concentrate Wilate and followed to assess clinical efficacy and tolerability. Dosing and monitoring were performed using FVIII assays. This is standard practice in most of the study sites and was feasible because of the product’s physiological 1:1 ratio and parallel PK profiles of FVIII and VWF. Dosing decisions were based on established guidelines and the investigators’ clinical judgment. The efficacy of the product was rated on a four-point scale by the investigator.

Results: Bleeding- 43 patients (25 of type 3) were treated for a total number of 1088 bleeding episodes (BE). The median dose per exposure day (ED) was calculated to be 31 IU VWF/FVIII:C/kg and were treated for a median of 1.2 days, with excellent or good efficacy achieved in 96% of treatments. Surgery- Efficacy and safety was also studied in 31 VWD patients who underwent 54 elective or urgent surgical procedures of which 27 were classified as major; the overall efficacy was rated as excellent or good in most cases (94%), with a mean dose per infusion being 34 IU VWF/FVIII:C/kg. Pediatric Use- A total of 8 children below 12 years of age with 310 BE s were treated, with an excellent/good efficacy in 98% of bleedings. Prophylaxis- 19 patients were on a prophylactic regimen for more than 10 consecutive weeks (total of 2,338 ED), with an overall reduction of bleeding frequency. Tolerability - Out of 5,662 rated infusions in all studies, the tolerability was assessed as “very good” or “good” in 99% of the cases.

Conclusions: The results of the prospective clinical trial program demonstrate the safety, efficacy and tolerability of this VWF/FVIII concentrate for the treatment of acute bleeding episodes, prophylaxis and surgical procedures in patients with VWD.
Tonsillar CD38+ Activated B Cell Fraction Harbours the Highest EBV Load in Patients with Infectious Mononucleosis.
Authors: Chaganti Sridhar , Bell Andrew , Heath Emily , Bergler Wolfgang , Kuo Michael , Starczynski Jane , Rudzki Zbigniew , Rickinson Alan
Published: October 2019

Epstein-Barr virus (EBV) persists within the B cell system and, in the blood of healthy virus carriers, is sequestered in the immunoglobulin (Ig)Dneg, CD27+ (“class-switched”) memory B cell compartment. This selective colonisation is apparent even in the blood of infectious mononucleosis (IM) patients undergoing primary EBV infection. Exactly how this is achieved is not understood, one theory being that the virus infects naïve B cells preferentially in vivo and drives them into memory using the physiologic process of germinal centre (GC) transit within lymphoid tissues. Here we have studied the distribution of EBV-infected B cells in tonsil cell preparations from acute IM patients and from chronic carriers. The following subsets of tonsillar B cells were isolated to high purity by FACS sorting and their EBV DNA loads were then determined by quantitative PCR: Naïve (CD38neg, IgD+, CD27neg); Class-switched memory (CD38neg, IgDneg, CD27+); Non-switched memory (CD38neg, IgD+, CD27+); Germinal centre (CD38+); and Plasma cell (CD38hi). In 15 carrier tonsils, the highest EBV load was consistently detected in class-switched memory, followed by significant amounts in non-switched memory. However, viral load in the CD38+ GC subset was usually very low, comparable to that in the naïve B cell fraction. This contrasted with the situation in 7 IM tonsils where overall viral loads were on average 100-fold higher than above. Here most viral DNA was consistently found in the CD38+ fraction, with lower loads in the class-switched memory and the smaller non-class switched memory subsets. We then investigated further the phenotype of the CD38+ population and found that, in IM tonsils, this not only included cells with the CD10 and CD77 markers of germinal centre origin but also other cells lacking these markers. Adopting a different FACS sorting strategy based on CD10, CD77 and IgD staining, we found that the virus was not present at significant levels in either the centrocyte (CD10+ CD77neg) or centroblast (CD77+) fractions. Thus EBV appeared to be concentrated in B cells expressing CD38 as a marker of activation rather than of germinal centre origin; this concurred with the results of immunohistochemical staining for the EBER RNAs showing an extrafollicular distribution of virus-infected cells in these IM tonsils. Our findings suggest that CD38 expression is a useful marker for B cells recently activated by EBV infection in vivo and show that, during primary EBV infection, the tonsillar CD38+ B cells lacking germinal centre markers harbour the highest viral load. If EBV does exploit the germinal centre pathway as an entry route into B cell memory, where primary infection is manifest as IM this pathway must be taken by a very small fraction of infected B cells and/or must have been completed before disease symptoms appear.
Complementary JAK/STAT Signalling Is Required for the Pro-Inflammatory Effects of CD40 Ligation: Differential Effects in Human Myeloid and B Cells.
Authors: Conzelmann Michael , Rodionova Elena , Hess Michael , Giese Thomas , Ho Anthony D. , Dreger Peter , Luft Thomas
Published: October 2019

CD40L represents a strong endogenous danger signal that induces pro-inflammatory activation of CD40-expressing cells such as dendritic cells (DC), monocytes, and B cells. However, since CD40 activation alone is insufficient to induce pro-inflammatory cytokines such as IL-12p70, we studied whether CD40-mediated pro-inflammatory activity might be dependent on co-signalling pathways involving JAK/STAT. Using quantitative Western blotting, we demonstrate that JAK/STAT signalling is induced by cytokines such as IL-4, GM-CSF and IFNg, whereas CD40 activation mediates NFkB signalling. CD40L-induced IL-12p70 and IL-10 secretion in human DC, monocytes, B cells, and chronic lymphocytic leukemia (CLL) cells was measured upon complementary JAK/STAT activation by IL-4, GM-CSF and IFNg in the presence and absence of specific inhibitors of JAK2, JAK3, and pan-JAK. Whereas IL-12p70 could not be induced by CD40 ligation or by cytokines alone, IL-12p70 secretion and suppression of IL-10 was reproducibly observed after co-stimulation of CD40L with IL-4, GM-CSF, or IFNg. This effect could be completely reversed by pan-JAK inhibition. Persistence of IL-4/GM-CSF/IFNg-mediated JAK/STAT signalling as late as 12 hours following cellular activation via CD40 was required for IL-12p70 secretion as shown by the effects of delayed JAK inhibition. Similarly, persistence between 12 and 24 hours of IL-12p35 and p40 mRNA expression correlated best with the level of IL-12p70 secretion. Specific inhibition of JAK2 and JAK3 further revealed a context-dependent action of the distinct JAK family members: JAK2 showed a strong co-dominant effect in the setting of IL-4-induced JAK/STAT activity. Both, JAK2 and JAK3 were required for IL-12p70 secretion, whereas JAK2 alone was sufficient to modulate IL-10 secretion. However, in the context of IFNg-induced JAK/STAT signalling in DC, neither JAK2 nor JAK3 inhibition had effects on IL-12p70. Here, only inhibition by the pan-JAK inhibitor involving JAK1 abrogated IL-12p70 secretion, indicating that in IFNg-dependent signalling, JAK2 is apparently sub-dominant to JAK1 and had only a small enhancing effect on IL-10. This context dependence markedly differed in myeloid cells and B cells, as normal and malignant (CLL) B cells maintain a co-dominant JAK2 activity in the context of IFNg-induced JAK/STAT-signalling. In conclusion, complementary JAK/STAT signalling is required for the pro-inflammatory effects of CD40 ligation in humans, with different JAK subset predominance in myeloid and B cells. These results may open new ways of lineage-specific interfering with CD40 signals by modulating JAK/STAT activity using tyrosine kinase inhibitors.
Acquisition of New In Vivo Genomic Change over Time in Multiple Myeloma.
Authors: Tai Yu-Tzu , Buon Leutz , King Benjamin , Li Cheng Li Cheng , Burger Peter Burger Peter , Li Xian-Feng , Song Weihua , Schlossman Robert , Richardson Paul , Anderson Kenneth C. , Munshi Nikhil
Published: October 2019

Multiple myeloma (MM) cells present with significant genetic abnormality. This genetic instability is considered responsible for not only development of malignant phenotype but also progression of myeloma as well as development of drug resistance. We have previously demonstrated that MM cell lines have elevated homologous recombination activity that leads to acquisition of new genomic changes over time and is associated with development of drug resistance (Blood2004; 104: 3409). However, such genomic evolution in patient samples has not been documented. Here, we have performed a genome wide loss of heterozygosity (LOH) assay, using high-density oligonucleotide arrays capable of measuring up to 500K single nucleotide polymorphisms (SNP) loci, and identify areas of amplification and losses to determine ongoing development of new changes that may reflect instability. We have evaluated 17 MM patients with purified myeloma cells obtained at two time points, at least six months apart. CD138–expressing myeloma cells from these patients were purified and their peripheral blood mononuclear cells were also obtained. Genomic DNA from these cells was digested with StyI, PCR amplified and hybridized to 250K SNP array. Results from CD138+ myeloma cells from two time points or more were compared with each other using the dChip software for LOH and copy number analysis. During the LOH analysis, we have adapted a tumor vs. tumor comparison to track all the new SNPs affected. We have observed that myeloma cells acquired new LOH loci in the subsequent samples. These new areas of genetic changes were recurrent; for example on chromosome 13, an average area of recurrent LOH of 87 Mb was found to be present among three patients. We were able to observe recurrence of significant copy changes on several chromosomes with/without LOH. The reproducible area of new acquisition of LOH and/or significant copy changes interestingly involved areas on chromosomes 1p, 1q, 8p, 9q, 13q, 15, 16q, 20p, 21q, and X that may have significant role in the pathogenesis and progression of the disease. We are currently analyzing the gene expression profile from these time points to identify expression changes correlating with the observed genomic changes on follow up samples. In summary we demonstrate continued acquisition of new genomic changes in vivo in MM over time and provide a molecular basis for evolution of more aggressive disease able to escape therapeutic interventions. The mechanism governing such evolution is an important target to not only inhibit progression of the disease but also development of drug resistance.