bluuebunny/crossref_metadata_2025 huggingface dataset:
Superior Survival after Autologous vs. Allogeneic Hematopoietic Stem Cell Transplantation (HCT) for Diffuse Large B-Cell Lymphoma (DLBCL) Not Explained by Differences in Chemosensitivity.
Authors: Hayes-Lattin Brandon , Carreras Jeanette , Zhang Mei-Jie , van Besien Koen , Vose Julie M. , Lazarus Hillard M. , Rizzo J. Douglas , Hari Parameswaran
Published: October 2019

No randomized trials have compared autologous HCT (autoHCT) to allogeneic HCT (alloHCT) for DLBCL. We analyzed the outcomes of 916 patients (pts) (837 autoHCT and 79 alloHCT) ages 18–60yrs after a first autoHCT or HLA-identical sibling alloHCT for DLBCL between 1995 and 2003 reported to the CIBMTR. Pts receiving T-cell depleted allografts or reduced-intensity conditioning were excluded. There were significant baseline differences between the groups in disease stage, B symptoms, extranodal disease and marrow involvement. AlloHCT pts were significantly more likely to have >3 chemo regimens prior to HCT (53 vs 40%), and resistant induction failure or relapse (39 vs 16%). At 1yr, treatment-related mortality (TRM) was higher after alloHCT (41%, 95% CI, 30–52%) than after autoHCT (11%, 95% CI, 9–14%, p<0.001), but risks of relapse/progression were similar (30%, 95% CI, 21–41%) and (33%, 95% CI, 29–36%, p=0.69), respectively. Cumulative incidence of outcomes and univariate probabilities of progression free (PFS) and overall survival (OS) at 5 yrs are summarized in table 1. In multivariate analysis, allo and autoHCT had differential early and late effects on outcomes. In the first 12 mo after transplant, alloHCT was associated with higher TRM (RR 4.76, 95% CI, 3.14–7.22, p<0.001), treatment failure (RR 2.08, 95% CI, 1.56–2.77, p<0.001) and mortality (RR 2.78, 95% CI, 2.06–3.77, p<0.001) but similar risk of progression (RR 1.14, 95% CI, 0.75–1.74, p=0.54) compared to autoHCT. Among pts surviving 12 mo post-transplant, no significant difference was observed between autoHCT and alloHCT for TRM, progression, PFS, or OS. Covariates that increased the risks of TRM and OS were older age (51–60 years), KPS <90%, chemoresistance at transplant, and earlier transplant yr (before 2001 vs later). Older age and chemoresistance were also associated with progression and lower PFS. There were no significant interactions between graft type and other prognostic variables; in particular, relative risk of outcomes with allo vs autoHCT were similar for patients with chemosensitive and chemoresistant disease. In summary, myeloablative alloHCT increased risks of early TRM and mortaliy without an effect on progression (compared to autoHCT). Transplant type did not affect outcomes after 12 months post-transplant. AutoHCT was associated with superior survival (fig 1), and the difference was not explained by differences in chemosensitivity at the time of transplant.

AutoHCT AlloHCT Outcomes (95%CI) (95%CI) AGVHD @ day 100 N/A 42 (31–53) CGVHD @ 5yrs N/A 27 (18–38) TRM @ 5yrs 18 (15–20) 45 (34–57) Relapse/progression @ 5yrs 39 (36–43) 33 (23–44) PFS @ 5yrs 43 (40–46) 26 (18–37) OS @ 5yrs 49 (46–53) 27 (18–27) Figure 1 Figure 1.
Front-Line Autologous Stem Cell Transplantation (ASCT) in Primary Mediastinal Large B-Cell Lymphoma: The GEL-TAMO Experience.
Authors: Rodriguez Jose , Conde Eulogio , Gutierrez Antonio , Garcia-Ruiz Juan Carlos , Lahuerta Juan Jose , Varela Maria Rosario , Perez-Equiza Encarnacion , Marin Julian , Albo Carmen , Caballero Maria Dolores
Published: October 2019

From 1985 to 2006, 71 patients with primary mediastinal large B-cell lymphoma receiving induction doxorubicine-based chemotherapy followed by ASCT as front-line therapy were registered in the GEL-TAMO (Spanish Group for Lymphoma and Autologous Transplantation). Median age was 28 years, 56% of patients were female, 40% presented with an ECOG ≥ 2; B-symptoms at diagnosis were present in 25% of the patients. Most patients presented with high-risk clinical features: bulky tumours defined as ≥10 cms of diameter were observed in most patients (87%), high LDH in 72% and, as previously reported (Rodriguez et al, Ann Oncol, 1994), β2m was elevated only in 7% of the cases. Forty-seven percent of patients presented 2–3 risk factors of the a-IPI. At transplant, thirty-five patients (49%) in first complete remission (CR), 23 (33%) in partial response and 13 patients (18%) failing the first induction therapy were transplanted, respectively. Conditioning regimens were BEAM or BEAC in 90% of the patients. 39 patients received Radiotherapy: 19 prior and 20 after the transplant. Most patients (91%) received peripheral stem cells. Only a patient failed to engraft after the transplant. After the transplant 73% of the patients achieved a CR and 17 patients were refractory. With a median follow-up from transplantation of 46,5 months the OS, PFS and DFS are 68%, 59% and 81% respectively. Progression of the disease was the main cause of death (78%). A patient died of a second neoplasia and 3 patients died of sepsis. There were no deaths related to transplant toxicity. By multivariate survival analysis both status of the disease at transplant (CR vs PR vs failure) and the Tumor score (Rodriguez et al, Ann Oncol,1992 ) were the only independent variables associated with the OS and PFS, respectively. In conclusion our experience, with a prolonged follow-up, shows that patients with primary mediastinal large B-cell lymphoma presenting at diagnosis with high-risk features have an encouraging survival and PFS with front-line ASCT. However, patients who received the transplant having failed the induction regimen have a very poor prognosis and should be tested with another innovative approach.
Deletion of the Short Arm of Chromosome 1 (del 1p) Is the Strongest Predictor of Poor Outcome in Myeloma Patients Undergoing an Autotransplant.
Authors: Ahmed Bilal , Saliba Rima M. , Ashraf Noman , Mendoza Floralyn , Hosing Chitra , Flosser Thuy , Weber Donna M. , Wang Michael , Couriel Daniel R. , Popat Uday , Kebriaei Partow , Alousi Amin M. , Champlin Richard E. , Giralt Sergio A. , Qazilbash Muzaffar H.
Published: October 2019

Background: Chromosomal abnormalities detected by conventional cytogenetic analysis are known to adversely impact the outcome in myeloma patients. Abnormalities of chromosome 1, 13, 14 and 17 are commonly described. Deletions of the short arm of chromosome 1 presumably result in a loss of tumor suppressor genes, including p73, a member of the p53 family. We report the outcome of 83 myeloma patients with clonal cytogenetic abnormalities who underwent high-dose therapy and single autologous stem cell transplantation at our institution.

Methods: We identified 83 patients (median age: 56 years) with clonal cytogenetic abnormalities detected at diagnosis by conventional chromosomal analysis. Patients underwent high-dose therapy and a single autologous transplant between April 2000 and May 2005. All 83 patients received high-dose melphalan, either alone (73) or as a combination of topotecan, melphalan and cyclophosphamide (TMC =10). Sixty-four patients were transplanted for the consolidation of first remission or for primary refractory disease, while 19 patients were transplanted for relapsed disease. The most common chromosomal abnormalities observed were hyperdiploidy (21%), del 1p (19%), 14q32 abnormalities (14%) and del 13q (10%).

Results: Median follow up among surviving patients was 25.5 months. Median interval from diagnosis to autotransplant was 7.7 months (range 2.5–52). Median progression-free survival (PFS) for the entire group was 19 months and the median overall survival (OS) was 52 months. On univariate analysis, both PFS and OS were significantly shorter in patients with del 1p (p= 0.001 and <0.0001, respectively), and those transplanted with relapsed disease (p= 0.03 and 0.04). Median PFS and OS for patient with del 1p were 12 and 22 months (vs. 26 moths and not reached for others), respectively. Thirty-two patients whose cytogenetic abnormalities resolved prior to autotransplant had a trend towards longer OS than the patients with persistent abnormalities (p = 0.08). Age, disease stage, β2m, serum albumin level, serum creatinine, and isolated abnormalities of chromosome 13q, 17 and 14 q32 did not emerge as significant predictors of outcome in this set of patients.

Conclusion: Del 1p is associated with a significantly shorter remission and survival in patients undergoing high-dose therapy and a single autologous transplant. Patients with this abnormality are candidates for novel therapeutic approaches including newer antimyeloma drugs and nonablative allogeneic transplantation.
CD3/CD19 Depleted Grafts for Haploidentical Stem Cell Transplantation in Children: Results of a Pilot Study.
Authors: Lang Peter J. , Mueller Ingo , Greill Johann , Bader Peter , Schumm Michael , Pfeiffer Matthias , Feuchtinger Tobias , Handgretinger Rupert
Published: October 2019

Profound depletion of T and B cells is a fundamental prerequisite for haploidentical transplantation. We present our preliminary results with a direct depletion procedure using antiCD3/antiCD19 coated magnetic microbeads and the CliniMACS™ device. Only patients with high risk malignancies (n=25, most of them had active disease or relapsed after previous trp.) or with nonmalignant diseases and high risk of graft failure (n=2) were included. The diagnoses were: AML/MDS/CML (n=13), ALL(n=6), relapsed Neuroblastoma/Ewing-/Rhabdomyosarcoma (n=6), SAA, PNH (n=2); Remission status: NR=8, 2nd transplantation=10, CR/CP1=3, CR2–4=6.

The patients received either TBI or Bu based standard conditioning regimens (n=9) or a toxicity reduced protocol (Flud, TT, Mel, OKT3, MMF, n=18). The grafts comprised a median number of 16x106/kg (7–41) stem cells as well as high amounts of NK-cells (137x106 (9–550)) and monocytes/granulocytes (6x108 (0.6–13)) with 49x103/kg (7–200) residual CD3+ cells and <0.01% CD20+ cells. Primary engraftment occurred in 89% of patients (after reconditioning and second stem cell donation:100%). Median time to reach >500 neutrophiles/μl and independence from platelet substitution was 10 and 9 days respectively. Recovery of CD3+ cells was favorable (d90: 320/-l, d180: 600/μl). Acute GvHD grade 0–1 occurred in 74%, 26% had GvHD grade II. Limited chronic GvHD occurred in 4 patients. No transplant related mortality (TRM) and, in particular, no lethal infections were observed. 13/27 patients (49%) were alive with a median follow up of 0.8 years (3 months – 2.1 years). Single cause of death was relapse. Disease status was predictive: 8/9 patients with leukemias and active disease relapsed, whereas only 2/10 patients in CR relapsed. 2/6 patients with solid tumors are alive.

Recovery of platelets was faster in patients with CD3/CD19 depleted grafts than in a historical control group of patients (n=53) transplanted with haploidentical positive CD34+ selected standard grafts (23 vs. 9 days, p<0.01). No lethal viral infections occurred in the study group due to the fast T cell recovery, whereas in patients with positive selected grafts a cumulative incidence of 18% was observed. The toxicity-reduced conditioning regimen helped to avoid any other TRM, despite intensive pretreatment (including previous transplantation) of the patients.

Conclusions: our preliminary results indicate that CD3/CD19 selected grafts can improve immunoreconstitution and TRM. Engraftment rates similar to that of patients with myeloablative standard conditioning regimens and positive selected stem cells could be achieved, although most patients of the study group received an intensity reduced regimen. However, the outcome was poor in patients with active disease. Thus, further options have to be investigated to increase the potential antileukemic activity of donor derived effector cells.
Favorable Outcome of Patients with Relapsed Hodgkin Lymphoma (HL) after Nonmyeloablative Hematopoietic Cell Transplantation (NM-HCT) Using Related Haploidentical Donors.
Authors: O’Donnell Paul , Luznik Leo , Symons Heather , Jones Richard , Gooley Ted , Harrington Elizabeth , Warnock Sandra , Sandmaier Brenda , Storb Rainer , Fuchs Ephraim
Published: October 2019

NM-HCT from related donors who share at least one HLA haplotype with the recipient is a treatment option for patients with hematologic malignancies who do not have suitable HLA-matched related or unrelated donors. We have shown that addition of cyclophosphamide pre-transplant (29 mg/kg) and post-transplant (50 mg/kg on day+3 or days+3 and +4) to a standard nonmyeloablative conditioning regimen of fludarabine and TBI combined with tacrolimus and MMF prophylaxis of GvHD is an effective means of achieving complete engraftment of donor T-cells and granulocytes with low non-relapse mortality and an incidence and severity of acute and chronic GvHD which does not differ from studies of NM-HCT using HLA-matched donors. In a study of 89 patients (FHCRC,N=30; JH,N=59) with advanced myeloid and lymphoid malignancies, patients with relapsed HL had significantly better survival than patients with other diagnoses. Fifteen patients with HL were studied (median follow-up of 17 mo). Patients were heavily pre-treated with a median number of prior cytotoxic therapy regimens of 5 (range: 3–10); 14 patients had failed prior autologous HCT with a median time of 18 mo from auto-HCT to NM-HCT. Donors for 9 patients were HLA-mismatched at ≥4/10 loci. All evaluable patients were complete donor CD3 and CD33 chimeras by day +28 (one patient died on day +28 and was not evaluable). Clinically significant acute GvHD occurred in 9/14 (64%) patients and Grades III/IV GvHD in 3/14 (21%) patients. Extensive chronic GvHD occurred in 5/14 (36%) patients. Two patients (13%) died of non-relapse causes at days +236 and +633 secondary to chronic GvHD. Median failure-free survival (FFS) was 21 mo compared to 6 mo for patients with other lymphoid malignancies (N=23) or myeloid malignancies [(N=51), see Figure]. The hazard of mortality was less among patients with HL compared to those with other lymphoid malignancies [hazard ratio (HR)=0.36 (p=0.05)] yet patients with myeloid malignancies had a similar hazard of mortality compared to those with lymphoid malignancies other than HL [HR=0.88 (p=0.66)]. Difference in FFS between HL and other lymphoid malignancies was not statistically significant [HR=0.54 (p=0.16)], nor was the difference between myeloid and other lymphoid malignancies [HR=1.26 (p=0.44)]. More patients will need to be studied to better demonstrate a graft-versus-lymphoma effect of haploidentical transplantation in relapsed HL. NM-HCT from haploidentical donors may be a good option for such patients who have a limited window of opportunity to proceed to transplant if responsive to salvage chemotherapy.

Figure Figure Figure Figure
Comparable Survival and Transplant-Related Mortality Following Allogeneic Hematopoietic Cell Transplantation from HLA Allele-Matched Unrelated and HLA-Identical Sibling Donors.
Authors: Cao Thai M. , Tsai Schickwann , Kelley Linda , Alder Stephen C. , Fuller Thomas C. , Pulsipher Michael A.
Published: October 2019

Comprehensive analyses of unrelated donor (URD) and recipient HLA-matching for allogeneic hematopoietic cell transplantation (AHCT) have demonstrated better outcomes when allele typing is performed using high-resolution nucleotide sequence-based techniques. To evaluate survival following myeloablative AHCT using allele-level HLA-matched URD as compared with HLA-identical sibling donors, we analyzed outcomes for 430 patients treated at our center between March 1991 and April 2005. Sequence-based allele typing was retrospectively performed for HLA-A, B, C, DR and DQ when not done at time of AHCT for URD (n = 124; 29%) and non-sibling related donors (n = 19; 4%). Donors were HLA-identical siblings (n = 276; 64%), HLA allele-matched URD (n = 52; 12%), single HLA-locus mismatched donors (n = 52; 12%), or > 1 locus mismatched donors (n = 50; 12%). The median age at transplant was 23.4 years (range: 0.2 – 61). The most common diagnoses were AML (n = 107; 25%), CML (n = 90; 21%), ALL (n = 86; 20%) and MDS (n = 50; 12%). Total body irradiation-based preparative regimens were used for 283 patients (66%). Bone marrow (BM) was the graft for 388 patients (90%) and GCSF-mobilized peripheral blood stem cells (PBSC) for the remaining 42 (10%). Graft-versus-host disease (GVHD) prophylaxes were cyclosporine and methotrexate (n = 327; 76%), long methotrexate (n = 42; 10%), T-cell depletion (n = 19; 4%), or other regimens (n = 42; 10%). With a median follow-up of 4.8 years (range: 0.2 – 12.1), the 5-year estimate of overall survival (OS) for the entire group was 48.2% (95% CI: 45.7 – 50.7) and transplant-related mortality (TRM) was 31.4% (95% CI: 28.8 – 34). As shown in the Table, OS and TRM were indistinguishable between AHCT performed with HLA-identical siblings compared with HLA allele-matched URD. There was also no difference in grade III – IV acute GVHD (P = .46) between these two groups whereas there was a trend towards more extensive chronic GVHD (HR 1.8; 95% CI: 0.9 – 3.6; P = 0.12) for the URD recipients. Using a multivariate analysis to adjust for advanced disease, age (> vs ≤ 30 years), graft (BM vs PBSC) and female-to-male gender mismatch, there remained no difference in OS between HLA-identical siblings and HLA allele-matched URD (P = 0.67). These results demonstrate that key outcomes (OS, TRM, and severe acute GVHD) are equivalent in recipients of grafts from either allele-level 10/10 HLA-matched URD or HLA-identical siblings.

Overall Survival TRM Number Hazard Ratio 95% CI P value Hazard Ratio95% CI P value HLA-ID Sibling 276 1 - - 1 - - HLA-ID URD 52 1.1 0.7 – 1.7 0.67 0.8 0.4 – 1.6 0.58 1 Locus MM 52 1.3 0.9 – 2.0 0.19 1.4 0.8 – 2.4 0.25 > 1 Locus MM 50 2.0 1.4 – 2.9 < 0.001 2.6 1.7 – 4.1 < 0.001
HLA Mismatched/Haploidentical Haematopoietic Stem Cell Transplantation for the Treatment of Chronic Myelogenous Leukemia.
Authors: Huang Xiao Jun , Xu Lan-ping , Liu Kai-yan , Liu Dai-hong , Chen Huan , Han Wei , Chen Yu-hong , Wang Jing-zhi , Chen Yao , Zhang Xiao-hui , Shi Hong-xia , Lu Dao-pei
Published: October 2019

Backgroud: Allogeneic marrow transplantation remains the only proven curative therapy for CML, but many patients have no suitably HLA matched related donor. Recently, we developed a new method for HLA-mismatched /haploidentical transplantation without in vitro T cell depletion. Conditioning including antithymocyte globulin followed by un-manipulated HLA-mismatched/haploidentical blood and marrow achieved comparable outcomes to HLA identical sibling transplantation. So good outcomes of HSCT from mismatched family donor for CML patients could be expected.

Methods: 81 consecutive patients received mismatche family donor HSCT for CML were analyzed. There are 40 cases in CP1, 10 cases in CP2 after BC or AP, 14 cases in AP and 17 cases in BC pre-HSCT. Patients received the modified BuCy2 regimen consisting of cytarabine, busulfan, cyclophosphamide, Me-CCNU and ATG. Both of donor G-BM (G-CSF immobilize ) and G-PB were harvested and infused to recipients. All patients were given CsA/MTX and MMF for prophylaxis of GVHD.

Results All patients achieved full donor chimerism. The cumulative incidence of aGVHD is 62.71% (CI 57.3– %−68.2%), grade III–IV is 24.69% (CI 28.7 %−30.7 %). 2 yrs cumulative incidence of cGVHD was 56.48 % (CI 49.6 %–−53.4%). 2 years OS is 71.33%(CI 66%–−76.7%), 2-year LFS were 61.23% (CI 54.66%–−67.8 %). The cumulative incidence of relapse is 17.74% (CI 11.3%–−14.2 %). The incidence of relapse in CML-CP1, CP2, AP and BC group pre-HSCT were 10.8% (CI 2.67%–18.95%), 0%, 9.2% (CI 0.5%–− 17.9%) and 42.71% (28.47%–56.95%%), with P=0.0150. The OS in CML-CP1, CP2, AP and BC group pre-HSCT were 67.66% (59.93%–75.39%), 83.33% (68.12%±098.54%), 77.38% (65.88%±88.90%), 69.68 % (58.32%±81.04 ) with p=0.7876.

Conclusion For patients with CML without an HLA identical sibling donor, haploidentical family members can be an alternative donor for HSCT. In our HSCT protocol, survival of HSCT for CML in advanced stage was no worse than that in stable stage. The optimal time of HSCT for CML patients from MM-family donors need to be further studied.
Assessment of the Immune Reconstitution by Cylex Immuknow™ Assay in Patients after Allogeneic Stem Cell Transplantation.
Authors: Gesundheit Benjamin , Azar Yehudit , Bringer Rachel , Steinberg-Prop Schivti , Israeli Moshe , Shapira Michael Y. , Resnick Igor , Samuel Simcha , Slavin Shimon , Or Reuven
Published: October 2019

After engraftment of allogeneic stem cell transplantation (allo-SCT) patients are severely immuno-suppressed and are susceptible for recurrent opportunistic infections on one hand; on the other hand, particularly without immunosuppressive treatment over-activity of the graft can lead to uncontrolled acute or chronic graft-versus-host disease (GvHD); optimal immunological activity of the graft is necessary to control the underlying malignant disease and to prevent relapse. Therefore, accurate immune monitoring is crucial for the assessment and appropriate management of the immune-compromised patients after allo-SCT. Since October 2005 up to 6 consecutive blood samples were collected from 51 patients undergoing allo-SCT in Hadassah Medical Center following engraftment. Immune function was analyzed by Cylex Immuknow™ assay, an FDA approved immune cell function test for the assessment of cell-mediated immunity. ATP activity from magnetically separated and lysed CD4 lymphocytes is measured by light intensity, thereby reflecting the current immune function of the patient. From other clinical applications of this test, the following stratification of the immune response was established according to the following levels of ATP activity: Low (0–225 ng/mL), moderate (225–525 ng/mL) and high activity (>525 ng/mL) of immune response. Our preliminary results from allo-SCT patients are in keeping with their clinical course and the known stratification of the ATP level:The gradual increase to moderate ATP levels over several months after allo-SCT are in keeping with normal immune reconstitution with an uneventful post transplant clinical course;high ATP levels were observed in patients prior to the clinical presentation of acute GVHD;low ATP levels were associated with recurrent infections and relapse.

Remarkably, there was no correlation with the recovery of the white cell count after allo-SCT. Our results with consecutive tests are the first study of Cylex Immuknow™ assay in patients after allo-SCT and are in keeping with other currently known clinical applications of this assay for other indications. Our preliminary observations indicate a promising contribution of Cylex Immuknow™ assay as a simple and fast monitoring technique for patients undergoing allo-SCT, which might also predict clinical complications. Our future follow-up studies aim to confirm these preliminary results in order to gain confidence in the assay’s clinical contribution as a possible standard test for patients after allo-SCT.
A Comparative Analysis of the Leukaemic Potential of Mature T Cells Versus T Cell Precursors.
Authors: Newrzela Sebastian , Baum Christopher , Li Zhixiong , Hansmann Martin-Leo , Hartmann Sylvia , Hartmann Marianne , Cornils Kerstin , Fehse Boris , von Laer Dorothee M.
Published: October 2019

After the report of two cases of leukaemia caused by insertional mutagenesis of a retroviral vector in children with SCID, it became clear that safety issues of therapeutic gene transfer must be addressed more thoroughly. We analysed whether gene transfer into mature T cells and haematopoietic stem cells bear the same risk of generating T cell leukaemia through activation of specific T cell oncogenes, such as LMO2, TCL1 and ΔTrkA. To address this issue, we used the Rag-1 mouse model, which allows long term analysis of transplanted T cells and haematopoietic stem cells. We were able to transduce mature T cells and haematopoietic stem cells of C57BL/6 (Ly5.1) donor mice with oncoretroviral vectors expressing LMO2, TCL1 and ΔTrkA. Transduction efficacies of up to 70% were achieved for mature T cells and approximately 90% for haematopoietic stem cells. After transplantation into Rag-1-deficient recipients, stem cell transplanted animals developed T cell lymphomas/leukemia for all investigated oncogenes after characteristic incubation times, mostly of a CD8+CD4+ double positive phenotype. T cell lymphomas were characterised by gross thymic mass, splenomegaly and heavily enlarged lymph nodes, although none of the control- vector- transduced mice developed lymphoma/leukaemia. LM PCR analysis revealed mono- or oligoclonality of the tumours. T cell transplanted animals showed no signs of leukaemia development so far. However, after several attempts, one immortalized T cell progenitor clone could be generated after transduction with LMO2. Our results so far indicate that mature T cells are less susceptible to transformation by known T cell proto-oncogenes, but the studies are still ongoing.
Intravenous Immunoglobulin (IVIG) Diminishes Immune-Mediated Clearance of Platelets Expressing an Integrin αIIbβ3 Transgene Product That Restores Hemostasis in a Canine Model for Glanzmann Thrombasthenia.
Authors: Fang Juan , Jensen Eric S. , Du Lily M. , Boudreaux Mary K. , Wilcox David A.
Published: October 2019

While individual inherited platelet disorders are rare, in aggregate, these diseases can account for a bleeding diathesis in ~1:20,000 individuals. One classic disease is Glanzmann thrombasthenia (GT), a rare autosomal-recessive bleeding disorder that is characterized by genetic defects of the platelet-specific integrin, αIIbβ3. Molecular abnormalities in either the αIIb- or β3-subunit can disrupt receptor synthesis, assembly, and/or function thereby preventing platelets from binding the receptors major adhesive ligands (von Willebrand factor and fibrinogen) to form platelet aggregates as a primary response to vascular injury. As a model to develop methods for gene therapy of platelet defects, GT is especially challenging because platelet integrin level as well as genetic transfer efficiency play a crucial role in restoring normal hemostasis. Developing effective treatment for animal models for this disease should ensure that a similar strategy would be effective for the treatment of other inherited platelet disorders. In experiments to be presented, SCF/G-CSF mobilized CD34+ peripheral blood cells (PBC) from integrin αIIb-deficient dogs were transduced with a lentivirus vector encoding the human αIIb integrin subunit driven by the tissue-specific αIIb promoter. GT dogs received an autologous transplant of lentivirus-transduced PBC using low dose (100–300 cGy) irradiation as transplant preconditioning and three scheduled doses of post-transplant in vivo drug treatment to enrich for transduced hematopoietic stem cells. Fluorescent immunocytometric analysis detected a moderate level (10%) of circulating blood platelets expressing αIIbβ3 following the initial transplant, which increased to 20–30% of normal levels following drug selection with O6BG and BCNU. Remarkably, we observed a significant improvement in a clinical sign of the GT phenotype with reduced bruising on the animals’ legs when αIIbβ3 was restored to the surface of only a fraction of circulating platelets. Further analysis showed restored ability of lentivirus-transduced platelets to aggregate and retract a fibrin clot in vitro using peripheral blood collected from these animals. Treatment of two animals with intravenous immunoglobulin effectively diminished platelet clearance due to an antibody response to newly expressed αIIbβ3 receptor. Reduced bleeding times were measured indicating the feasibility for targeting platelets with genetic therapies for better management of patients with inherited bleeding disorders. These studies should help us to define molecular and cellular events necessary for targeting therapeutics to platelets, which will contribute to eventual, safe, effective, and technologically viable strategies for correcting inherited bleeding disorders in humans.
In Utero Transplantation of Transduced Hematopoietic Stem Cells Results in Deletion of Transgene-Specific T Cells.
Authors: Laje Pablo , Peranteau William H. , Endo Masayuki , Zoltick Philip W. , Flake Alan W.
Published: October 2019

The developing fetal immune system provides a unique opportunity to manipulate normal immunologic development for therapeutic prenatal and anticipated postnatal interventions. In previous studies we have shown that allogeneic in utero hematopoietic cell transplantation (IUHCT) results in donor specific tolerance that can subsequently facilitate non-myeloablative postnatal cellular or organ transplants. It follows that in utero injection of transduced hematopoietic stem cells (HSC) could potentially induce tolerance to a transgene encoded protein. We hypothesized that expression of a transduced antigenic protein by HSC and their progeny would alter thymic T cell development resulting in deletion of antigen specific T-cells. To test this hypothesis, we used the mammary tumor virus (MTV) superantigen system to evaluate the effect of IUHCT of transduced HSC on T cell development. In this system, expression of different MTV oncogenes by different I-E+ strains of mice results in deletion of T cells expressing the relevant Vβ T cell receptor. Specifically, mice which are Mtv7+ delete T cells expressing the Vβ6 T-cell receptor. In this study, CD150+CD48− enriched Balb/c (I-E+ Mtv7−) HSC were transduced with an HIV-based lentivirus expressing MTV7 under an MND promoter. 1.5E+05 transduced cells were injected intravascularly via the vitelline vein into E14 Balb/c fetuses. Non-injected age matched naive Balb/c mice served as the control group. The peripheral blood (PB) and thymuses of injected fetuses and control mice were harvested at day of life (DOL) 10, 20 and 60 and analyzed by flow cytometry for T lymphocyte Vβ6 expression. Additionally, the T cell composition of the thymus was assessed at DOL10 for CD4 and CD8 single positive (SP) and CD4/CD8 double positive (DP) cells. Thymic flow cytometric analysis at DOL10 revealed that greater than 98% of the T cells were CD4CD8 DP, a stage that has not yet undergone negative selection. No significant difference was noted in the percentage of thymic Vβ6+ DP T-cells at this time point or at DOL20 and DOL60. In contrast, there was a significant decrease in the percentage of Vβ6+ peripheral blood SP cells in those mice injected with MTV7 transduced HSC relative to control mice at DOL10, DOL20 and DOL60 (p<0.05) (Fig 1). The current study supports the ability of enriched transduced HSC to induce deletion of transgene specific T cells after IUHCT. In the future, this strategy may be useful to promote tolerance for pre or postnatal cellular or gene therapy.

Figure Figure
Disparity in Diagnosis of Pulmonary Embolism in Hospitalized Patients with Pneumonia.
Authors: Kapoor Avina , Dang Qianyu , Ragni Margaret V.
Published: October 2019

Pulmonary embolism (PE) is a major cause of preventable hospital deaths. Little is known about PE in the setting of pneumonia, as symptoms and signs of PE and pneumonia overlap, confounding diagnosis. With infrequent thromboprophylaxis, PE continues to be a major public health problem. In order to determine the incidence and characteristics of PE in patients with pneumonia, we analyzed statewide discharge data, 1997–2001, collected by the Pennsylvania Health Care Cost Containment Council (PHC4). The analysis included all PE discharges with ICD-9 codes 415.11 and 415.19, excluding pregnant patients and all second or later PE admissions. Infectious disease diagnoses were enumerated by ICD-9 codes, including 030–041, 042, 790.7, 996–998; pneumonia by ICD-9 codes 480–483. 485–487, and 996–998; and diagnostic procedures by revenue code 352 for computerized tomographic (CT), including spiral CT scans. Severity of illness was determined by MediQual Altas Admission Severity Categories. Of 33,036 total PE discharges (all-PE) from Pennsylvania hospital discharges 1997–2001, 5,757 (16.8%) had infection, more than half of which, 3,543 (52.3%), were due to pneumonia (pneumonia-PE). There was a marginal increase in the incidence of pneumonia-PE during this period, 615 (10.3%) in 1997 to 839 (10.5%), p>0.05. Those with pneumonia-PE were typically Caucasian, 77.3%, p<0.01; female, 51.5%, p<0.01; and elderly, 53.1% ≥ 70 years of age, p<0.01, mean age, 66.4±17.3 yr. The proportion of patients undergoing CT scans increased by 15.3%, from 18.9% in 1997 to 21.8% in 2001, p<0.01, an increase 20% lower than than the increase in the all-PE group, 88.9%, from 10.8% in 1997 to 20.4% in 2001, p<0.01. There was a significantly smaller improvement in the severity of illness scores in the pneumonia-PE group, increasing (46.0% to 53.0%) for the three lowest Altas categories and decreasing (46.8% to 42.0%) for the two highest categories, 1997–2001, respectively, compared with the all-PE group, (56.9% to 68.7%) in the three lowest and (35.3% to 26.5%) for the two highest categories, both p<0.01. During this period, mortality rates decreased significantly less in the pneumonia-PE group, by 18.5%, from (21.1% to 17.2%) as compared to a 20% decrease in all-PE, from (11.5% to 9.2%), 1997–2001, p<0.01. The apparent disparity in CT scan frequency and associated lesser improvements in severity of illness and mortality in the pneumonia-PE group, as compared with the all-PE group, suggest early PE diagnosis may have been missed. Modeling the rate of increase in PE diagnosis per increase in CT scan in the all-PE group, we estimate 454 missed cases of PE, which is 12.8% more PE cases among those with pneumonia. We conclude that the lack of increase in CT scans among hospitalized patients with pneumonia is associated with lower rates of PE diagnoses and poorer outcomes. Thromboprophylaxis should be considered in hospitalized pneumonia patients, particularly those at risk for PE.

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Purification of a Single Peptide with Vitamin K Epoxide to Vitamin K and Vitamin K to Vitamin KH2 Activity.
Authors: Chu Pei-hsuan , Huang Teng-yi , Williams Jason , Stafford Darrel W.
Published: October 2019

More than 21 million prescriptions for warfarin are written yearly in the US. Yet, in spite of its importance, vitamin K epoxide reductase (VKOR), the target of warfarin, has resisted purification since its identification in 1972. We report the first successful purification and reconstitution of activity of a recombinant human vitamin K epoxide reductase. A series of detergents were screened to determine that best for solubilization of VKOR from microsomes. Detergents tested that were effective in solubilization of VKOR also led to loss of measurable activity. This loss of activity supports our previous prediction that VKOR is embedded in and requires a membrane environment for enzymatic activity. The short-chain phospholipid, DHPC (1,2-Dihexanoyl-sn-Glycero-3-Phosphocholine) was the detergent of choice to efficiently extract VKOR from the microsomes, even though this reagent completely inhibited enzyme activity. Partial reconstitution was achieved on-column by washing with 0.4 % dioleoylphosphatidylcholine/0.4% deoxycholate. Complete recovery of activity was achieved by removing the deoxycholate through dialysis in the presence of the reducing reagent, THP (Tris(hydroxypropyl)phosphine). During dialysis, the solution became cloudy indicating the formation of membrane-like structure. Purified recombinant VKOR is ~21 kDa (~18.5 kDa + tag); fully active; and over 93% pure. The concentration of warfarin for 50% inhibition is the same for purified protein and microsomes. It has been reported and assumed that VKOR is a multi-subunit enzyme. Our results, however, suggest that a single peptide can accomplish the reaction. The trace amounts of contaminating proteins were identified by mass spectrometry; however, none are apparently relevant to the VKOR reaction. Moreover, the turn-over number of purified VKOR (0.25 sec-1 is approximately two-fold higher than microsomes and about 10 fold higher than the turnover number of gamma-glutamyl carboxylase for CO2 addition. In addition to the vitamin K epoxide to vitamin K reaction, our results also indicate that VKOR can efficiently convert vitamin K to vitamin K epoxide. Our results suggest that ancillary proteins (other than a thioredoxin-like enzyme) are not necessary for full VKOR activity. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination.
Concordance with National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines (CPGs): A Quality of Care Tool for Newly Diagnosed Patients (pts) with Mantle Cell Lymphoma (MCL).
Authors: Rodriguez Maria Alma , Veer Anna Ter , Nademanee Auayporn , Niland Joyce , Lepisto Eva , Kho Michelle , LaCasce Ann , Weeks Jane Carrie , Czuczman Myron , Millenson Michael , Friedberg Jonathan , Zelenetz Andrew D.
Published: October 2019

Introduction: In 1999, the NCCN published its first NHL CPG and in 2000, established the NCCN NHL Outcomes Database Project to monitor patterns of care, CPG concordance and outcomes in participating institutions. We report here on clinical characteristics and CPG concordance among newly diagnosed (dx) pts with MCL in the database.

Methods: We prospectively collected demographic, staging, and treatment information on consecutive pts with MCL presenting at 5 geographically diverse NCCN institutions (Dana-Farber, Roswell Park, City of Hope, Fox Chase and MD Anderson). We assessed concordance with 2 CPG’s relevant to the concordance impacting the majority of pts: 1) bone marrow biopsy (BMBx) as part of the initial work-up and 2) use of an endorsed first line regimen among pts with stage III/IV disease. CPG concordance was assessed by comparing each pt’s care against the version on the NCCN guideline in effect at the time the pt was diagnosed.

Results: Between 7/2000 and 10/2005, we enrolled 132 MC evaluable pts. Median age was 58; 43% had high-intermediate or high risk disease according to the IPI at presentation; 123 (93%) pts presented with stage III/IV disease. The median follow-up was 22.6 months. Overall, 91% of pts underwent a staging BMBx as recommended by the guidelines. Concordance varied by institution, low 78% to high 98%. Among 123 pts with stage III/IV disease, first-line therapy was concordant with CPG recommendations in only 59%. Use of rituximab accounted for 92% of all non-concordance. When the guidelines were modified in 2003 to include rituximab as an option for first-line therapy of MCL, concordance rose from 31% (2000) to 100% (2003–5). Of concordant pts receiving combination chemotherapy, 33% received CHOP-based standard dose therapy and 62% received dose-intense therapy. NCCN guidelines consider all therapy administered as part of a clinical trial to be concordant; trial-directed treatment accounted for 42% of concordant care.

Conclusions: Our data suggest that the majority of MCL pts in these centers receive care that is concordant with current standards. In this subgroup of patients, participation in clinical trials occurred at an impressively high rate. However, not all pts undergo BMBx as a routine component of staging as recommended by NCCN guidelines suggesting that this is an area for potential quality improvement. This study also highlights that differences in management exist even within national comprehensive cancer centers. Because long-term follow-up is possible with this database, future studies will assess the initial treatment and guideline concordance on long-term outcomes in this unique group of pts.
Quality of Life and Satisfaction with Iron Chelation Therapy in Patients with Beta Thalassemia Major: Results from the ITHACA Study.
Authors: Mantovani Lorenzo Giovanni , Scalone Luciana , Ravera Simona , Rofail Diana , Cappellini Maria Domenica ,   the ITHACA Study Group
Published: October 2019

Background Patients with beta Thalassemia Major (TM) require life-long blood transfusions, which often cause iron overload that may increase patients’ morbidity and mortality. Iron Chelation Treatment (ICT), aimed to reduce iron overload, is based on 8–12 hour infusions of Deferoxamine (DFO) for 5–7 days/week, and/or Deferiprone (L1) orally administered. Current ICT can be related to low satisfaction, low compliance, and potentially negative consequences on clinical effectiveness, patients’ wellbeing and on healthcare costs.

Aims: To investigate the Health-Related Quality-of-Life (HRQoL) of TM patients and their satisfaction with ICT.

Methods: The Italian-THAlassemia-Cost-&-Outcomes-Assessment (ITHACA) was a naturalistic multicentre study conducted to evaluate costs, HRQoL, compliance and treatment satisfaction in TM patients undergoing ICT for at least 3 years, enrolled at Italian Thalassemia Care Centers. HRQoL was measured in >14 years old patients with 2 generic instruments: EQ-5D; Short Form-36 (SF-36). To measure satisfaction >12 years old patients received a 28-item instrument consisting of 4 domains: ‘perceived effectiveness’, ‘acceptance’, ‘burden’, and ‘side effects’. Each domain scored from from 1 (very dissatisfied) to 5 (very satisfied). Results Based on 126 patients: median age 29.4 years (12.3–48.5), 49.6% male. At enrolment 48.0% were using DFO, 33.6% L1, 18.4% were treated with DFO+L1. 86.5% of patients had at least one TM-related complication, 13.5% changed treatment regimen at least once in a median of 11.6 months before enrolment. With EQ-5D profile patients reported moderate problems with ‘mobility’ (9.1%), ‘self care’ (0.8%), ‘usual activities’ (23.5%), moderate or severe ‘pain/discomfort’ (60.5%) and ‘anxiety/depression’ (39.5%). Mean EQ-5D-Visual Analogue Scale was 73.0 (30–100). The SF-36 Physical Component Summary mean(SD) score was 47.7(8.4), while the mean score estimated in the Italian general population comparable for age and sex is 53.3; the Mental Component Summary mean(SD) score in TM patients was 45.1(8.8), while the Italian general population mean score was 47.7. Mean satisfaction scores were 4.29 (perceived effectiveness), 3.37 (acceptance), 3.87 (burden), and 3.57 (side effects). Simple linear regression analysis showed that satisfaction with burden (r2=12.6%, p<0.0001), side effects (r2=12.3%, p<0.0001) and acceptance(r2=11.3%, p=0.0001), are positively associated with the adherence item ‘never thinking about stopping medication’ (p<0.0001 in both cases).

Conclusions: TM patients on ICT have impaired levels of physical and mental HRQoL. Therapies improving patients’ satisfaction and compliance to ICT may have positive consequences not only on clinical effectiveness but also on overall patients’ well-being.
Long-Term Follow-Up of Treatment for Acute Myeloid Leukemia with Allogeneic Hematopoetic Cell Transplantation Using BuCy2 as Preparation.
Authors: Pant Shubham , Crilley Pamela Ann , Dodds Anthony J. , Szer Jeffrey , Bolwell Brian J. , Elder Patrick , Styler Michael , Kalaycio Matt , Sobecks Ronald , Nivison-Smith Ian , Avalos Belinda R. , Copelan Edward A.
Published: October 2019

In 1991, we reported the results of 127 patients with Acute Myeloid Leukemia (AML) who underwent conditioning for allogeneic transplant with busulfan, 4 mg/kg on each of 4 days and cyclophosphamide 60 mg/kg on each of 2 days. In the current abstract, we report data on 295 patients, including the initial cohort, who underwent allogenic transplantation for AML from 1984 to 1995 at 7 participating institutions. Median age of patients is 33. One hundred forty nine men and 126 women underwent transplant. All patients received marrow from related HLA-identical donors. One hundred seven patients (36%) remain alive. One hundred forty one patients were transplanted in first remission (group I), 43 in second remission (group II) and 111 patients were in > 2nd remission or had refractory disease (group III). The median follow-up of patients in the initial study was 3 years and is 12 years in the current study. Seventy-five percent of patients surviving 3 years after transplantation are estimated survivors 12 years after transplant. Relapse (20), chronic GVHD (5) and infection (3) were the major causes of death in patients who died more than 3 years after transplantation. The estimated relapse rate for group I at 3 and 12 years is 18%(95% CI: 2–32%) and 29% (95% CI 11–47%) for group II is 40% (95% CI: 8–72%) and 52% (95% CI: 16–84%), and group III is 59% (95% CI: 37–81%) and 69% (95% CI: 45–93%) respectively. The estimated leukemia-free survival (LFS) for group I at 3 and 12 years is 60% (95% CI: 44–76%) and 47% (95% CI: 29–65%), for group II is 47% (95% CI: 17–77%) and 34% (95% CI: 4– 64%)and group III is 22% (95% CI: 6–38%) and 15% (95% CI: 1–29%) respectively. Remission stage was predictive of long-term LFS (P<0.001), with patients transplanted in first remission doing better than the rest. Older age was a risk factor for late failure (death or relapse beyond 3 years, P=0.05). Remission stage at transplant (p=0.44), chronic GVHD (P=0.08) and acute GVHD (P=0.12) were not significantly predictive of late failure. In conclusion a vast majority of patients alive and well at 3 years post transplant seem destined to be cured. A proportion of patients do fail beyond 3 years, but the remission stage of disease at transplant is not predictive of late failure.
Pediatric Hematopoietic Progenitor Cell Collection and Transplant: Does Pegfilgrastim Represent Progress?.
Authors: Merlin Etienne , Auvrignon Anne , Dubray Claude , Rapatel Chantal , Deméocq François , Kanold Justyna
Published: October 2019

The particular technical challenges of apheresis in children require the highest quality of stem cell mobilisation. To date, G-CSF remains the reference. The arrival of pegfilgrastim (pegf) theoretically allows the maintenance of an elevated and constant serum level of G-CSF after only one injection.

DESIGN: eleven children (2–16 yrs) undergoing chemotherapy for malignancy were consecutively included in a prospective pilot study. Mobilization consisted of one sc injection of 300 μg/kg pegf in haematological steady state. Apheresis was begun once the value of 20 CD34+/μl cells was reached in the blood, or at day 4 after pegf administration.

RESULTS: at first apheresis the mean CD34+ cell, mononuclear cell and polynuclear blood counts were respectively 98/μl (12–353), 7.26×109/l (1.0–9.5) and 39.3×109/l (4.6–57.3). The CD34+cell peak level was obtained on day 3 after injection (3–5). The first standard apheresis (less than 3 patient blood volumes) allowed the harvest of more than 5×106 CD34+ cells in 9/11 pts, the median CD34+ cell harvest being 13.5×106/kg (range 1.6–36). On day six after injection, the median ANC was 13.6×109/l. These results were compared to 3 historical prospective cohorts of children who received non-conjugated G-CSF at 10, 20 or 2×12 μg/kg/j, respectively. The four cohorts were not different for age, weight, diagnosis and rounds of chemotherapy. Mobilisation with pegf was more efficient than with G-CSF at 10 μg/kg/d (p<0.001); there were no significant differences between the other regimens.

Figure Figure

Tolerance: 8 pts had an elevation of LDH, 3 a hyperuricemia respectively up to 2 times and 1.5 times the upper limit. After reinfusion, polynuclear recovery (ANC>500/μl) was obtained after (median) 11.5 days (11–13); platelet recovery (>20 G/l) was reached on median day 17 (12–20).

CONCLUSION: the combination of predictable mobilisation kinetics similar to those of non-pegylated G-CSF, a better acceptance profile together with an efficiency which appears to be at least as good, seems to represent progress in stem cell mobilisation in children. However, sustained blood WBC levels indicating that hematopoiesis has not returned to steady state necessitates a slight delay (at least 7 days) in resuming the chemotherapy program.