bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-Term Dosing of AMG 531 in Thrombocytopenic Patients with Immune Thrombocytopenic Purpura: 2-Year Update.
Authors: Bussel James B. , Kuter D.J. , de Wolf J.Th.M , Guthrie T.H. , Newland A. , Wasser J.S. , Hamburg S.I. , Bjorkquist L. , Guo M. , Nichol J.
Published: October 2019

AMG 531, a novel thrombopoiesis-stimulating peptibody, increases platelet production by stimulating the TPO receptor. This study is an ongoing, open-label extension assessing the safety and efficacy of long-term, weekly, SC administration of AMG 531 in patients with chronic ITP who completed a previous AMG 531 study. Patients previously treated with AMG 531 received the same starting dose as the final dose given in the previous study; placebo-treated patients began the extension at 1μg/kg. Doses could be adjusted based on platelet response. As of an April 2007 analysis, 137 patients were enrolled and 136 had received AMG 531. The longest treatment duration was 122 weeks; 22 patients were followed for 96 weeks or longer. At baseline, 91 women and 46 men had a mean age of 53±15(SD) years and a median (range) platelet count of 18(1–50)x109/L; 82 patients (60%) had undergone splenectomy. The most frequently reported adverse events (AEs) were headache (overall incidence 31%), contusion (27%), fatigue (24%), diarrhea (24%), epistaxis (23%), nasopharyngitis (21%), and arthralgia (20%). Exposure-adjusted analysis showed no trend for AEs to increase in frequency with increased drug exposure (table). Eleven patients had serious AEs judged by investigators as treatment-related including 3 withdrawn from study (vaginal hemorrhage, increased reticulin in the bone marrow reported as myelofibrosis, and initial report of multiple myeloma later reclassified as monoclonal gammopathy of undetermined significance - 1 report each) and 8 who continued on-study (bone marrow disorder/reticulin fibrosis - 3, unacceptably high platelet count - 2, thrombosis - 2, and cerebral thrombosis/papilloedema/temporary blindness - 1). One patient developed neutralizing antibodies to AMG 531 (absent on retesting 4 months after drug cessation), with no clinical sequelae and no cross-reactive antibodies to TPO. Overall, 112 patients (82%) achieved a platelet response (≥50x109/L and doubling of baseline). From week 4 onward, weekly incidence of platelet response ranged from 52–73% (figure). Median number of weeks to first response was 2 (median dose 3μg/kg). Of 30 patients with baseline use of concurrent ITP therapies, 13 were able to discontinue them and 6 additional patients had a >25% dose reduction. Individualized weekly doses of AMG 531 provide a therapeutic option for long-term treatment of chronic ITP. The safety profile has been acceptable, and most patients have been able to maintain a platelet response and discontinue or reduce concurrent ITP therapies.

Exposure-Adjusted incidence of AEs with Overall Incidence ≥ 20% Platelet Response (≥ 50x109/L and Doubling of Baseline) by Study Week Platelet Response (≥ 50x109/L and Doubling of Baseline) by Study Week
Genome-Wide Association Study for Hb F Determinants in Sardinian Patients with Thalassemia Major and Intermedia.
Authors: Anni Franco , Galanello Renzo , Surrey Saul , Fortina Paolo M. , Bowser Mark , Devoto Marcella
Published: October 2019

Expression of fetal globin is silenced normally in adult life; however, determinants linked and/or unlinked to the globin-gene clusters could modify Hb F expression so it persists into adults. Increased expression in adults offers hope as a cure for sickle cell disease (SCD) and beta thalassemia, since formation of FS hybrids in SCD inhibits deoxy Hb S polymerization while increased fetal chain expression compensates partially for decreased adult beta-globin chains in beta thalassemia. Characterization and controlled manipulation of high Hb F determinants is critical to decreasing clinical severity of these life-threatening genetic diseases, which result in high morbidity and mortality worldwide. We report on analysis of a unique beta-thalassemia cohort from Sardinia who present with either

a mild, non-transfusion-dependent (NTD) form expressing high Hb F, or with a severe, transfusion-dependent (TD) form expressing low Hb F.

Both groups are homozygous for the beta39 chain-termination mutation and lack adult beta globin. Genome-wide DNA arrays were run on 42 TD and 33 NTD patients using the Affymetrix 500K (500,568 SNPs in a total of 23 individuals) or 6.0 (909,622 SNPs in a total of 52 individuals) SNP chip platforms. The average sample call rates were 96.3% for the 6.0 chip and 94.3% for the 500K chip. Data from the two chips were merged and a total of 482,243 SNPs that were present in both chips were considered for genetic association analysis using a case/control design (NTD versus TD). Quality control filters removed 42,166 SNPs (8.7%) with genotype call rates < 90%. An additional 54,683 SNPs (12.4%) were removed because they either failed a test of Hardy-Weinberg equilibrium at a p< 0.0001 or had a minor allele frequency (MAF) of < 0.01. The genotype call rate for the remaining 385,394 SNPs was 97.3%. Preliminary analysis of difference in allele frequency distributions in the two groups of patients did not reveal any signal of association that remained statistically significant when corrected for multiple tests. The smallest p-value observed was 2x10-6 for SNP rs8028098 on chromosome 15. However, several SNPs in two candidate regions on chromosomes 2p16 and 6q23 were nominally significant (p<0.05). The smallest p-values observed in these two regions were 0.004 for rs11886868 located in the BCL11A gene, and 0.002 for rs210950 in the MYB gene. These results confirm the likely involvement of these two genes in the regulation of Hb F expression, and show the usefulness of the association approach in the search for genetic modifiers of Mendelian disease phenotypes. Additional samples are being analyzed in an attempt to achieve sufficient power to reach genome-wide significance.
Salvage Allogeneic Hematopoietic Cell Transplantation with Fludarabine and Total Body Irradiation (3 or 4 Gy) after Failure of First Allografts.
Authors: Gyurkocza Boglarka , Cao Thai M. , Storb Rainer F. , Lange Thoralf , Leisenring Wendy , Franke Georg , Sorror Mohamed L. , Maloney David G. , Shizuru Judith , Sandmaier Brenda M.
Published: October 2019

We analyzed data from 38 patients (median age = 56, range: 8 – 68 years) with acute leukemia (n=15), chronic idiopathic myelofibrosis (n=6), myelodysplastic syndrome with or without myeloproliferative disorder (n=5), chronic myeloid leukemia (n=4), non- Hodgkin lymphoma (n=4), aplastic anemia (n=2), multiple myeloma (n=1) and renal cell carcinoma (n=1), who underwent salvage allogeneic hematopoietic cell transplantation (HCT) for allograft failure. In 14 cases the original donors were used for second HCT, while in 24 cases different donors were identified (Table 1). Conditioning regimens for first HCTs included total body irradiation (TBI; 2 Gy) with or without fludarabine (Flu; n=28), myeloablative regimens (busulfan-cyclophosphamide, n=6; cyclophosphamide-TBI, n=2); and other, cyclophosphamide-anti-thymocyte globulin-based regimens (n=3). Conditioning for salvage HCT consisted of Flu 30 mg/m2/day on days -4 to -2 followed by TBI of 3 (n=24) or 4 (n=14) Gy on day 0. Cyclosporine and mycophenolate mofetil were used for postgrafting immunosuppression. The median time between first and salvage HCTs was 91 (range, 29 to 1004) days. Sustained second grafts were achieved in 34 patients (89%), while grafts failed in 4 patients (11%), all of whom had idiopathic myelofibrosis. With a median follow-up among surviving patients of 2.0 (range, 0.3 to 7.8) years, the 2 and 4 year Kaplan-Meier survival estimates were 49% (95% CI: 31%, 66%) and 42% (95% CI: 23%, 61%), respectively. The 2 year relapse-rate and non-relapse mortality were 36% (95% CI: 20%, 52%) and 25% (95% CI: 11%, 41%), respectively. The cumulative incidences of grades 2–4 acute and moderate-severe chronic graft-versus-host disease (GVHD) at 2 years were 42% and 41%, respectively. Four patients with chronic GVHD discontinued systemic immunosuppressive therapy at a median of 2.5 years. Within the limitations of the small patient numbers studied, TBI dose (3 vs. 4 Gy), same vs. different donors for salvage HCT, donor type (related, unrelated, HLA-haploidentical related vs. double umbilical cord), and HCT comorbidity scores did not appear to affect outcomes. Based on this retrospective multicenter analysis, we conclude that graft failure following allogeneic HCT can be effectively overcome by second transplantation using conditioning with Flu and low dose TBI (3 or 4 Gy), which should be further investigated in a prospective manner.

Table 1. Donors in 1st and 2nd HCTs. HLA-MURD: HLA-matched unrelated donor; HLA-MMURD: HLA-mismatched unrelated donor, UCB: umbilical cord blood.

2nd HCT Different Donor 1st HCT Same Donor HLA-MURD HLA-MMURD Double UCB HLA-haploidentical HLA-identical sibling 11 11 - - - - HLA-MURD 17 3 10 4 - - HLA-MMURD 8 - 1 7 - - Double UCB 2 - - - 1 1
Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloblastic Leukemia (AML) or Myelodysplastic Syndrome (MDS) in Patients (pts) Older Than 50 Years: a Retrospective Single Center Study
Authors: Robin Marie , Porcher Raphael , Ades Lionel , Raffoux Emmanuel , de Latour Régis Peffault , Petropoulou Anna , Boissel Nicolas , Ribaud Patricia , Devergie Agnès , Rocha Vanderson , Fenaux Pierre , Dombret Hervé , Socié Gérard
Published: October 2019

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment in MDS, and also in many cases of AML, 2 diseases where median age is higher than 50 years.

Aim: Analyse the outcome of patients older than 50 years who received an allogeneic hematopoietic stem cell transplantation (HSCT) for MDS or AML, in Saint-Louis Hospital from January 1997 to December 2007.

Method: 48 patients (pts) aged from 50 to 68 years (median: 56) received an HSCT for MDS (N=28) or AML (N=20) during this period. AML pts had intermediate (n=15) or high risk cytogenetics (n=3). 8 pts had failure to initial induction chemotherapy. FAB classification for MDS was: RAEB (n=8), RAEBt (n=6), secondary AML (n=2) or refractory anemia (n=2). Maximal IPSS score was high, intermediate-2 and intermediate-1 in 11, 14 and 4 pts. Ten pts with MDS received an “AML-like” chemotherapy and 5 received demethylating agents before transplant. 18 of the AML pts were in complete remission (CR) at time of transplant (CR1, n=15; CR2, n=3) and 2 were in relapse at time of HSCT. 10, 9 and 9 patients with MDS had < 5%, 5–10% and > 10% blasts in bone marrow at time of HSCT.

Results: 19 patients with AML (95%) and 16 patients with MDS (57%) received a HSCT from an HLA-identical sibling donor. Other pts received a matched unrelated donor. Thirty-two pts, including 9/20 and 23/28 had at least one co-morbidity according to Sorror score. The conditioning regimen was myeloablative (MAC) in 14 pts (29%). Reduced intensity (RIC) was fludarabine based in 34 pts, associated with either 2 Gy TBI (n=25) or chemotherapy (n=9). All pts engrafted. 35 pts had acute graft-versus-host disease (GVHD): grade I in 11, grade II in 18 and grade III in 6. GVHD incidence did not differ between pts who received a MAC or a RIC regimen. Two-year overall survival (OS) was 41% (95% CI: 26–57). OS and relapse-free-survival (RFS) were similar after MAC or RIC regimen (OS: 29% vs 35% and RFS: 29% vs 30%). Short-term non-relapse mortality (NRM) was lower in patients who received a RIC as compared to patients who received a MAC regimen but was similar at long-term (6-month NRM = 21% vs 9% and 12-month NRM = 21 vs 19 %). Patients with AML or MDS had similar OS and RFS (OS: 42 vs 41%; RFS: 37 vs 26% at 2 years for AML and MDS, respectively). NRM was not significantly higher in pts with MDS (26 vs 10% at one year) whereas relapse rate was not significantly higher in pts with AML (13 vs 6%).

Conclusion: HSCT for AML or MDS after 50 years is a curative option for pts with related or unrelated HLA-identical donor regardless kind of conditioning regimen and co-morbidity at time of transplantation.
Incorporating Marrow Plasma Cell Infiltration at Diagnosis and Cytogenetic Features into Prognostic Scoring at Point of Autologous Stem Cell Transplantation for Multiple Myeloma
Authors: Klammer Matthias , Giles Chrissy , Reid Alistair G , Ross Fiona M , Szydlo Richard M. , Pavlu Jiri , Apperley Jane F. , Rahemtulla Amin
Published: October 2019

240 patients (152 male, 88 female) with a median age of 59 years (27ys–73ys), referred to Hammersmith Hospital, London, for high dose therapy (HDT) and autologous stem cell transplantation (ASCT) between 1994 and 2007 were treated in a uniform fashion with myeloablative doses of Melfalan. The median OS post HDT/ASCT of all patients was 53 months with a median time to disease progression (TTP) of 20 months. In univariate analysis (194 pts) a > 60% plasmacell infiltration of trephine biopsy at presentation was associated with a significantly shorter median TTP (15 months) following HDT/ASCT compared to patients with a lesser degree of marrow plasmocytosis, whose median TTP was 20 months (p=0.003). Studies of chromosomal aberrations were undertaken by metaphase cytogenetics and interphase FISH in 142 pts at the time of ASCT and yielded interpretable results in 96 (67%) pts. Failure of conventional G –banding and FISH was universally due to low plasma cell content of the marrow aspirate below 5% of nucleated cells. 59 pts (42%) did not show any chromosomal abnormalities at point of transplantation; 37 pts (26%) had chromosomal aberrations, involving the IgH gene locus on chromosome 14q32 and/or deletions of the long arm of chromosome 13 (del 13q) in 33 cases. Deletion of the short arm of Chromosome 17 (del 17p) was only seen in one patient. In multivariate analysis (92 pts), independent risk factors for early relapse post ASCT were progressive disease/minimal treatment response at time of transplantation (RR 5.95, 95%CI 2.86–12.57), a plasma cell infiltrate of more than 60% on marrow trephine at diagnosis (RR 3.4, 95% CI 1.68–6.88) and presence of cytogenetic abnormalities other than sole t(11;14) or del 13q (RR 2.04, 95% CI 1.0–4.17) at time of ASCT. An additive scoring model based on these predictive factors (Table 1) identifies a low risk group of 49 pts (score 0) with a median progression free survival (PFS) of 33 months, an intermediate risk group of 34 pts (scores 1–2) with a median PFS of 15.7 months and a high risk group of 9 pts (scores 3–4), whose median PFS is 4.6 months (p<0.0001).

Table 1: Prognostic scoring model

Risk Factor Score Plasmacells >60% on diagnosis trephine biopsy 1 Plasmacells <60% 0 Progressive disease/minimal response at time of ASCT 2 Complete response/partial response 0 Normal Cytogenetics or t (11.14)/del 13q as sole abnormalities at time of ASCT 0 All other chromosomal aberrations 1

FIG.1: Progression free survival in low risk (score 0), intermediate risk (1–2) and high risk (3–4) patients FIG.1:. Progression free survival in low risk (score 0), intermediate risk (1–2) and high risk (3–4) patients
Outcome of African-American (AA) Multiple Myeloma (MM) Patients after Autologous Stem Cell Transplantation (ASCT): A Single-Center Experience.
Authors: Wu Yin , Goloubeva Olga , Ruehle Kathleen , Yanovich Saul , Rapoport Aaron P. , Akpek Gorgun , Badros Ashraf Z.
Published: October 2019

The incidence of MM in African-Americans is more than double that in Caucasians. Historically AAs have had a higher mortality rate than Caucasians; but over the past 10 yrs, the age-adjusted mortality rate has been on the decline for AAs while it has been stable for Caucasians as a result of ASCT and novel agents. Previous studies (n=74 AA patients) suggested that response to ASCT is similar, if not better, for AA patients (Verma et al 2008, Saraf et al 2006). We retrospectively analyzed the clinical presentation of a large cohort of AA patients (n=103) who underwent ASCT at our center between 1998 and 2008 and compared their outcome to that of Caucasians patients (n=183) transplanted in the same time period. AA patients were significantly younger than Caucasian patients at diagnosis with median age 53 (range: 32–75) vs 59 (range: 27–80), respectively (p<0.0001). The distribution of isotype and stage of MM at diagnosis were similar between the two groups. Approximately 20% patients in each group presented with renal insufficiency. Among the AA patients, 45% had albumin < 3.5 g/dL; 14% and 18% had beta2-microglobulin > 3.5 mg/L and > 5.5 mg/L, respectively. Initial cytogenetic data were not available for the majority of patients. Median time from diagnosis to ASCT was significantly longer for AA than for Caucasian patients at 0.8 yrs (range: 0.23–9.2) vs 0.5 yrs (range: 0.1– 7.0), respectively (p<0.0001). There was no difference in incidence of transplant-related complications, as reflected by similar lengths of hospital stay, with a median of 15 days for both groups. No significant difference in response to ASCT was found between the two groups. Median EFS was 1.7 yrs (range: 1.5–2.7) for AAs and 1.8 yrs (range: 1.5–2.4) for Caucasians (p=NS). At a median follow up of 5 yrs, 74% of AA and 70% of Caucasian patients were alive. Median OS was also not significantly different at 9 yrs (range: 6.7- not reached) for AAs and 8 yrs (range: 5.9-not reached) for Caucasians (p=NS). Cox regression model for prognostic markers: albumin, calcium, creatinine, hemoglobin, and platelet count were significant for OS in AA patients (p range 0.004–0.0001). The current study is the largest one of AA patients undergoing SCT at a single institution. AA patients with MM present at a younger age, but undergo ASCT at a significantly later time from diagnosis than Caucasian patients. This delay may reflect a disparity in referral pattern and access to health care. AA patients have similar EFS and OS after ASCT. It is likely that better responses to the newer anti-myeloma agents, as well as favorable impact of ASCT, even if delayed, may explain the improvement of AA patients with MM over the past decade. Further studies of responses of AA patients to novel myeloma agents are needed.
Sequential VAD (Vincristine, Adriamycin, Dexamethasone) and VTD (Bortezomib, Thalidomide, Dexamethasone) Induction Followed by High-Dose Therapy with Autologous Stem Cell Transplantation and Maintenance Treatment with Bortezomib for Newly Diagnosed Multiple Myeloma: Final Analysis of Phase II Trial.
Authors: Yoon Sung-Soo , Kim Hye Jin , Chung Joo Seop , Eom HyeonSeok , Jang Jun-Ho , Kang Hye Jin , Kim Cheol Soo , Kim Kihyun , Lee Dongsoon , Lee Jae Hoon , Lee Junglim , Lee Sang Jae , Min Yoo Hong , Seong Chu-Myong , Sohn Sang-Kyun , Suh Cheolwon , Won Jong-Ho
Published: October 2019

Background: Incorporation of novel agents has resulted in improved response rate and reduced side effects in multiple myeloma. Especially in newly diagnosed multiple myeloma, novel agents are very promising as induction chemotherapy.

Methods: Patients are planned to receive 2 cycles of VAD (vincristine 0.4mg D1–4, adriamycin 9mg/m2 D1– 4, dexamethasone 40mg D1–4, 9–12 every 3 weeks), and VTD (bortezomib 1.3mg/m2 D1, 4, 8, 11, thalidomide 100mg daily, dexamethasone 40mg D1–4, 9–12 every 3 weeks). High dose melphalan (200mg/m2) is used as a conditioning regimen for ASCT. Bortezomib (1.3mg/m2) as a maintenance treatment is administered weekly x 4 times every 6 weeks for 4 cycles after ASCT. Response was assessed by EBMT criteria, with additional category of nCR. Adverse events were graded by the NCI-CTCAE, Version 3.0.

Results: Total 71 patients were enrolled, and efficacy could be assessed in 62 patients. After 2 cycles of VAD, response rate was 71% (95% CI, 59.7–82.3). After VTD, overall response rate was more increased to 96% (95% CI, 90.8–100, rate of CR and nCR 27%). Especially, nine patients with poor prognostic cytogenetics all showed response after VTD. So far, autologous stem cells were successfully collected in 42 patients with a median CD34+ count of 6.49 x 106/kg (range, 0.6–44.7 x 106/kg) except one patient. After autologous stem cell transplantation, twenty five patients completed bortezomib maintenance, and the rate of CR + nCR was 68%. The median follow-up duration was 16.4 months, and median time to response was 1.6 months. Median time to progression was not reached, and 1 year survival rate was 98%. In total, 127 cycles of VAD and 115 cycles of VTD were given and grade 3,4 hematologic toxicity was more frequently observed after VAD than VTD (anemia 18.1% vs 8.7%, neutropenia 12.6% vs 4.3%), and incidence of grade 3 peripheral neuropathy after VAD was lower (1.5%) than VTD (7%). All patients received low-dose aspirin prophylaxis. Deep vein thrombosis was observed in two patients, but this was not related with thalidomide.

Conclusions: Sequential VAD and VTD induction therapy in newly diagnosed multiple myeloma was very effective, even in patients with poor prognostic cytogenetics, and did not prejudice stem cell collection. VTD as induction therapy has contributed to increased response rate and decreased side effects. *Protocol Number : KMM51-NCT00378755
Defect in DNA Double Strand Break Response in Paediatric ALL Is Caused by Upregulation of Multiple Pro-Survival Pathways That Can Serve as Therapeutic Targets.
Authors: Stankovic Tatjana , Marston Eliot , Weston Victoria , Agathanggelou Angelo , Mapp Katie , Kearns Pamela , Falciani Francesco , Moss Paul , Taylor Malcolm
Published: October 2019

The molecular basis of clinical heterogeneity in paediatric ALL is poorly understood. Defects in cellular responses to DNA double strand breaks (DSBs) have been associated with resistance to DNA damaging agents and poor clinical response in patients with leukaemia. We have previously shown that one third of paediatric ALL tumours exhibit in vitro defect in ionising radiation (IR)-induced apoptosis, despite retained integrity of p53 pathway. In this study we addressed molecular basis of defective (DSB) response in paediatric ALL with the view of identifying novel therapeutic targets.

We analysed differences in transcriptional and posttranscriptional in vitro responses to IR in 22 ALL patients, stratified as either apoptotic resistant or apoptosis sensitive. Analysis of coordinate IR induced expression of functionally related genes, by two independent methods (gene set enrichment analysis, GSEA, and functional module analysis) revealed in resistant cases differential upregulation of multiple pro-survival pathways involved in EGF, PDGF, IGF, PI3 and MAPK signalling. In response to IR resistant ALLs also showed differentially induced phosphorylation of Akt and S6, downstream targets in PI-3 pathway. Array analysis of 42 receptor tyrosine kinases (TRKs), revealed differential phosphorylation of TRKs that act upstream of the PI3 including ErbB3,4, FGF R4,2a, VEGF R1,2 as well as IGF-I R. Furthermore, we were able to demonstrate that in apoptotic resistant leukaemias PI3/Akt upregulation during IR response lead to differential phosphorylation of DNA-PK catalytic subunit, a protein involved in non homologous end joining (NHEJ) repair of DNA DSBs. Consequently, increased repair of DSBs was observed in apoptosis resistant ALL cases and this was documented by faster resolution of IR induced intranuclear foci of γH2AX, a marker of DNA DSBs. Finally, pharmacological inhibition of pro-survival pathways PI3-Akt, IGF and MAPK in resistant leukaemias restored sensitivity to IR.

We suggest that abnormal activation of pro-survival pathways during DSB response may represent one of the mechanisms of resistance to DNA damaging agents in paediatric ALLs and that targeting these pathways should be considered as therapeutic approach in aggressive form of disease.
Evolution of Gene Expression Signatures in Relapsed Childhood Acute Lymphoblastic Leukemia Differs Based on Timing of Relapse.
Authors: Bhojwani Deepa , Wang Jinhua , Yang Jun J , Morrison Debra , Devidas Meenakshi , Raetz Elizabeth , Hunger Stephen P. , Relling Mary V. , Carroll William L.
Published: October 2019

The outcome for childhood acute lymphoblastic leukemia (ALL) following marrow relapse remains bleak in spite of numerous approaches to further intensify therapy. Understanding the biological basis of relapse and chemoresistance, as well as identifying and validating potential new targets, are the goals of our study. Previously we determined global gene expression patterns of matched diagnosis and relapse leukemic blasts in 32 patients (64 samples) with childhood B-precursor ALL using Affymetrix U133A arrays (Blood2006;108(2):711–7). We now have extended this analysis to 60 patients (120 samples). Thirty-six patients relapsed early (within 36 months of initial diagnosis), while 24 patients relapsed late. Within the TEL/AML1 subset (n=12 patients), time to relapse was inversely proportional to the correlation co-efficient of expression profiles of the diagnosis and relapse matched pair samples, suggesting that the later the relapse, the more distinct the relapse clone is from the diagnostic clone. A supervised pairwise analysis in all 60 patients identified 292 probesets that were differentially expressed between diagnosis and relapse (FDR < 10%). In a relative enrichment analysis, multiple genes mediating cell death were down-regulated at relapse (p=0.00003), suggesting that the leukemia cells had evolved mechanisms to enhance survival. These included p21, TNFPAI3, RIPK2, BCLAF1, STK17B. In concert, DNA replication genes were up-regulated at relapse (p=0.00002). Differences in pathways leading to early vs. late relapse were evident. Early relapse was characterized by an over-expression of cell cycle genes reflecting a proliferative state. At the time of relapse, a marked over-representation of genes involved in the progression through the M phase of the cell cycle was observed in early relapse compared to late relapse (p=1.3E-08). Late relapse was characterized by the over-expression of genes involved in nucleoside biosynthesis, particularly targets of antifolates (DHFR, MTHFD1, TYMS). A small number of gene expression patterns were common to both early and late relapse, including up-regulation at relapse of BIRC5 (survivin): an attractive target for therapeutic intervention. In conclusion, analysis of an expanded cohort of matched diagnosis/relapse pairs has validated and extended our previous findings that early relapse is associated with a proliferative gene expression signature. In addition we have now identified pathways operative in late relapse. Targeting these individual genes and pathways may lead to innovative strategies to treat or prevent relapsed ALL.
Transforming Activity of AML1-ETO Oncoprotein Is Independent of CBFβ and ETO but Requires Homo-Oligomeric Interaction.
Authors: Zeisig Bernd B , Kwok Colin , Qiu Jihui , Dong Shuo , So Chi Wai Eric
Published: October 2019

AML1/RUNX1 and CBFb encode two critical transcription factors essential for the generation of hematopoietic stem cells (HSC). In acute myeloid leukemia, where leukemic stem cells (LSCs) have been functionally identified, AML1 and CBFb also represent the most commonly mutated targets. While animal models indicate that AML1 fusions per se are not sufficient to induce full-blown leukemia, they enhance self-renewal and expand targeted HSC and early progenitors, a property also reported for other oncogenic transcription factors involved in acute leukemia. Although attempts had been made to identify the critical domains required for AML-ETO (AE) mediated transformation, conflicting results were presented from most of these studies using exploited wellestablished cell lines, which suffer from the pitfall of carrying irrelevant genetic aberrants that may not reflect the normal biology of the disease. The only available structure/ function data on primary cells was limited to NHR2 of the ETO portion of the fusion, but it does not distinguish the functional contribution between homo-oligomerization and hetero-oligomerization. The lack of comprehensive structure/function data has not only significantly impeded the progress of understanding the biology of the disease, but also hinders the development of specific therapeutics. To this end, we performed extensive functional analysis to identify the key components essential for AE-mediated transformation of primary hematopoietic cells. In spite of the critical role of CBFb for wild type AML1 functions and its direct involvement in chromosomal translocation, we demonstrate that multiple AE single point mutants defective in CBFb interaction were still capable of transforming primary hematopoietic cells. Consistently, shRNA mediated knockdown of the endogenous expression of CBFb in primary cells did not compromise the transforming activity of AE, strongly suggesting a dispensable function of CBFb in AE mediated transformation. On the other hand, we demonstrate that NHR2 as the only domain in the ETO portion of the fusion is essential for transformation, but its heterooligomeric function including interaction with transcriptional repressor ETO family proteins is dispensable for the transforming activity. In contrast, synthetic FKBP homooligomerization modules could functionally replace NHR2, indicating that AE mediated transformation is critically dependent on homo-oligomeric property of the resultant fusion. Moreover, the transformation can also be abolished by a small molecule inhibitor that specifically dissociates homo-oligomerization. Together, these results not only identify the essential components and refine potential avenues for therapeutic targeting of AE oncogenic complexes, but also strongly endorse a common homo-oligomerization dependent mechanism shared by the most prevalent leukemia associated transcription factors.
Identification of MicroRNA Markers of Sensitivity to the Novel Bruton’s Tyrosine Kinase (BTK) Inhibitor PCI-32765 in Non-Hodgkin’s Lymphoma.
Authors: Balasubramanian Sriram , Chen Jun , Honigberg Lee , Tee Lillian , Mamuszka Hannah
Published: October 2019

PCI-32765 is a novel, first-in-class small molecule inhibitor of Bruton’s tyrosine kinase (BTK), currently in preclinical development and shortly entering Phase I clinical trials for the treatment of hematological malignancies. BTK is essential in B-cells for signalling via the B-cell receptor (BCR), and mutations in BTK have been shown to cause x-linked agammaglobulinemia in humans and x-linked immunodeficiency (xid) in mice. We have found that PCI-32765 has demostrated significant growth inhibitory activity in vitro in a number of non-Hodgkin’s lymphoma (NHL) cell lines as well as primary human NHL tumors cultivated ex-vivo. However, certain subsets of NHL cell lines were found to respond better to the inhibitor, and in keeping with this, a wide variance was also observed in the response among primary human tumors. PCI-32765 had GI50 <3uM in 7/15 cell lines (46%), including 3 follicular lymphoma (FL) and 3 diffuse large B-cell lymphoma (DLBCL) lines. Based on these results, we tested primary tumors including 15 FL and 7 DLBCL, utilizing a modified version of Oncotech’s EDR Assay. We found that while only 1 of the DLBCL tumors showed a similar response, PCI-32765 had a response rate of 40% in the primary FL samples. The reasons for this variation are still under investigation but one reason may be that that not all tumors are equally dependent on BTK activation for their survival. In order to obtain a predictive marker for response to PCI-32765 in human tumors, we utilized a microRNA (miRNA) profiling approach in the primary FL tumors using a commercially available miRNA array platform. We hypothesized that miRNA might be a better diagnostic tool than mRNA because miRNA function is more conserved and it is more stable than mRNA (thus easier to harvest successfully from paraffin embedded blocks). Further, it has been shown that miRNA signatures can predict responsive and resistant phenotypes based on basal miRNA levels, and have been found to have essential functions in lymphomas. When the miRNA profiles from the FL tumors were analyzed, we found that a signature consisting of 4 miRNAs could predict the sensitive tumors with a p-value <0.01. Of these, 2 were upregulated and 2 were downregulated in the sensitive samples compared to the resistant ones. It is likely therefore that these are implicated in the mechansisms of sensitivity and resistance to PCI-32765 in the FL tumors, and the connections between genes regulated by these miRNAs to pathways controlled by BTK are being explored.
Complications of Hysterectomy in Women with Von Willebrand Disease.
Authors: James Andra H , Myers Evan R , Cook Chad , Pietrobon Ricardo
Published: October 2019

Background: Case reports and small case series suggest that women with von Willebrand disease (VWD) are at a very high risk of bleeding complications with hysterectomy. Because the procedure may be beneficial to women who suffer from heavy menstrual bleeding, an understanding of the true risks involved is essential for appropriate decision making.

Objectives: To estimate the incidence of bleeding and other complications in women with VWD who undergo hysterectomy.

Methods: The United States Nationwide Inpatient Sample (NIS) from the Healthcare Cost and Utilization Project of the Agency for Healthcare Research and Quality for the years 1988–2004 was queried for all hysterectomies for nonmalignant conditions. Data were analyzed based on the NIS sampling design. Bivariate analyses were used to examine the differences between women with and without VWD. Multivariate analysis was used to adjust for potential confounders among women who underwent hysterectomy for heavy menstrual bleeding.

Results: 545 of the 1,358,133 hysterectomies were to women with VWD. Women with VWD were significantly more likely to experience intraoperative and postoperative bleeding (2.75% versus 0.89%, p < 0.001) and require transfusion (7.34% versus 2.13%, p < 0.001) than women without VWD. One woman with VWD died (odds ratio = 28.49).

Conclusions: While the risk of bleeding complications from hysterectomy in women with VWD is smaller than previously reported, women with VWD did experience significantly more bleeding complications than women without VWD. Nonetheless, for women who have completed childbearing, the risks of hysterectomy may be acceptable.
Excluding HIT Diagnosis by a Particle Gel Immunoassay.
Authors: Schallmoser Katharina , Drexler Camilla , Rohde Eva , Strunk Dirk , Groselj-Strele Andrea , Lanzer Gerhard , Kroll Hartmut , Panzer Simon
Published: October 2019

Heparin-induced thrombocytopenia (HIT) is a prothrombotic condition, characterized by a platelet drop of >50% and/or thrombosis with a temporal relationship of 1–2 weeks after initiation of heparin treatment. These surrogate markers are useful for the clinical assessment but are hardly applicable in multi-morbid patients with clinical conditions mimicking HIT. Platelet activation assays (heparin-induced platelet aggregation assay, HIPA; serotonin release assay, SRA) and platelet factor-4/polyanion enzyme immunoassays (PF4-ELISA) confirm HIT. HIPA and SRA are highly specific but laborious requiring selected donor platelets and extended experience. High titer IgG antibodies correlate with clinical HIT, but ELISA is also time-consuming. The alternative H/PF4-antigen particle gel immunoassay (ID-PaGIA H/PF4®) is easy, provides results within one hour and detects mainly IgG, but also IgA/M antibodies. We evaluated specificity and sensitivity of the PaGIA in comparison to HIPA and ELISA (PF4-ENHANCED®) in 285 patients (median 71yrs, range 1–97, 45% cardiovascular surgery; f/m 56/73, and 55% medical, f/m 78/78) with undetermined likelihood for HIT and 89 healthy controls (median 42yrs, range 26–64, f/m 49/40) to validate it as a rapid assay to exclude HIT. HIPA was positive in 12% of patients. Based on ROC curves (ELISAs vs HIPA), OD-cutoff values for the IgG/A/M- and IgG-ELISA were 0.761 and 0.564, respectively. In controls the OD of IgG-ELISA was 0.066 (0.028–0.500) and lower than from patients’ samples negative by PaGIA and IgG/A/MELISA (n=158; 0.074; 0.009–0.339; p=0.017). PaGIA was positive in 70 patients (25%). In both patient groups both ELISA ODs were higher if PaGIA and IgG/A/MELISA were positive compared to negative PaGIA but positive IgG/A/MELISA (p<0.001; data are shown as median; range). Analyzed by ROC curves (ELISAs vs HIPA), IgG/A/MELISA (OD-cutoff 0.761) and IgG-ELISA (OD-cutoff 0.564) had sensitivities of 81% and 86%, and specificities of 75% and 81%. Positive and negative predictive values were 31% and 97% (IgG/A/MELISA) and 39% and 98% (IgG-ELISA), respectively. PaGIA, IgG/A/M- and IgG-ELISA were significant predictors for the results of HIPA (p<0.001) and the IgG-ELISA had the highest explained variance (41%). Based on our results the PaGIA may serve as a rapid test to exclude HIT since a negative PaGIA is rarely associated with high titer IgG PF4-antibodies or a positive HIPA. In cases of positive PaGIA results and in patients highly suspicious for HIT but with negative PaGIA, alternative anticoagulation should be considered until results from functional testing are available.
Platelet-Targeted Expression of Coagulation Factor VIII (FVIII) Shows Efficacy for Using the Dog as a Large Animal Model for Gene Therapy of Hemophilia a
Authors: Wilcox David A. , Du Lily M. , Haberichter Sandra L. , Jacobi Paula M. , Fang Juan , Jensen Eric S. , Shi Qizhen , Montgomery Robert R.
Published: October 2019

In order to develop a large animal model for platelet-targeted gene therapy of Hemophilia A, we transduced normal canine bone marrow with a lentivirus vector under the transcriptional control of integrin αIIb gene promoter driving expression of human BDD-FVIII. Bone marrow was collected from normal dogs and transduced in the presence of recombinant canine c-SCF, c-IL-6, human h-TPO, and h-flt-3/flk-2 ligand differentiation factor. Immunofluorescence analysis using antibodies against human FVIII, and the megakaryocyte-specific marker integrin αIIb revealed synthesis of FVIII within tissue cultured megakaryocytes derived from lentivirus transduced bone marrow cells. This result appeared similar to synthesis, trafficking and storage of FVIII trafficked to α-granules of the murine “small animal” model for hemophilia A and human megakaryoyctes in vitro. To examine the effect of FVIII expression in platelets, in vivo, FVIII-transduced canine bone marrow was xenotransplanted into immune-compromised “NOD-SCID” mice treated with a sublethal dose (350 cGy) of irradiation. Flow cytometric analysis using antibodies specific for canine glycoproteins Ibα and integrin αIibβ3 revealed that circulating canine platelets comprised approximately 20–30% of the total platelet population in whole blood isolated from the mice within four weeks after xenotransplant. Immunofluorescence confocal microscopy detected a punctuate staining for FVIII that co-localized with a platelet α–granule protein, P-selectin, within a subpopulation of canine platelets isolated from murine whole blood. In contrast, FVIII was not detected in platelets from control samples. In addition, chromogenic analysis of platelets isolated from mice transplanted with FVIII-transduced bone marrow demonstrated the presence of a biologically active form of FVIII, FVIII:C at 2 mU/ml/lysate of 1×108 platelets. These results indicate that canine megakaryocytes are able to synthesize and store a biologically active form of FVIII that can be retained within progeny platelets. We speculate that dogs affected with hemophilia A should serve as an ideal “large animal” model to test if FVIII can undergo regulated release from platelets following physiologic hemostatic response to vessel injury. This raises the possibility of developing a locally inducible secretory pool of FVIII in platelets of patients with Hemophilia A following autologous transplantation of FVIII-transduced hematopoietic stem cells.
Directed Evolution of Immune-Escaping Adeno-Associated Virus Vectors
Authors: Maersch Stephan , Huber Anke , Hallek Michael , Buening Hildegard , Perabo Luca
Published: October 2019

Efficiency of therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity against the natural virus. Genetic engineering by rational design or directed evolution has been employed in the last 3 years to generate capsids that escape antibody neutralization and has led to identify several amino acid residues of the capsid proteins that can be mutated in order to decrease antibody recognition (Perabo et al., 2006; Maheshri et al, 2006; Lochrie et al., 2006). In this novel study, we aimed to exploit the comprehensive knowledge gathered so far by generating novel capsid variants that carried multiple point mutations at these previously identified sites. Capsid libraries were generated by codon randomization of several immunogenic residues and screened to isolate mutants that most efficiently infected human cells despite the presence of anti-AAV2 neutralizing antibodies. Besides testing novel combinations of concomitant mutations at these sites, this approach allowed for the first time an exhaustive scanning of combinations of all 20 natural amino acids at each position. We identified several novel capsid mutants that remain highly infectious even when incubated with serum concentrations that completely neutralize wild type AAV2. Our results demonstrate that combining mutations at several sites it is possible to improve the immune-escaping ability of the capsid. In addition, we show that escaping ability and other biological characteristics of these mutants are strongly dependent on the type of amino acid substituted, demonstrating that an exact choice of substituted amino acids is essential to maximize stealth properties and minimize loss of packaging ability, particle stability and transduction efficacy. These vectors can be used for therapeutic gene transfer to patients with pre-existing immunity, or for repeated treatment after antibodies are generated upon first application.
Production of High Titer cGMP-Grade SIN Gamma-Retroviral Vectors by Transfection in a Closed System Bioreactor
Authors: Van der Loo Johannes C.M. , Swaney William , Nordling Diana , Schambach Axel , Baum Christopher , Williams David A. , Reeves Lilith , Malik Punam
Published: October 2019

The need for gamma-retroviral vectors with self-inactivating (SIN) long terminal repeats for clinical trials has prompted a shift in the method with which large scale GMP-grade vectors are produced, from the use of stable producer lines to transient transfection-based techniques. The main challenge of instituting this methodology was to develop SIN retrovirus vectors that produced high amounts of genomic vector RNA in packaging cells, and to design scalable processes for closed system culture, transfection and virus harvest. Using improved expression plasmids, the Vector Production Facility, an academic GMP manufacturing laboratory that is part of the Translational Cores at Cincinnati Children’s Hospital, has developed such a method based on the Wave Bioreactor® production platform. In brief, cells from a certified 293T master cell bank are expanded, mixed with transfection reagents, and pumped into a 2, 10 or 20 Liter Wave Cell Bag containing FibraCel® discs. Cells are cultured in DMEM with GlutaMax® and 10% FBS at 37°C, 5% CO2 at a rocking speed of 22 rpm and 6° angle. At 16–20 hrs post-transfection, the media is changed; virus is harvested at 12-hour intervals, filtered through a leukocyte reduction filter, aliquoted into Cryocyte freezing containers, and frozen at or below −70°C. Several processing parameters, including the confluency of cells harvested prior to transfection, the timing of transfection, the amount of plasmid DNA, exposure of cells to PBS/TrypLESelect, and the timing of the media change post-transfection affected vector titer. Mixing cells with plasmid and transfection mixture prior to seeding onto FibraCel, as compared to transfecting cells 1-day post-seeding (as is standard when using tissue culture plastic) increased the titer from 104 to 4 × 105 IU/mL. Similarly, increasing the amount of plasmid DNA per mL from 4.6 to 9.2 μg doubled the titer in the Wave, while it reduced titer by 20–40% in tissue culture flasks (Fig. 1). Using an optimized protocol, six cGMP-grade SIN gamma-retroviral vectors have now been produced in support of the FDA’s National Toxicology Program (NTP), with unconcentrated vector titers ranging from 1 × 106 to as high as 4 × 107 IU/mL. Using similar processing, we have produced a large scale SIN gamma-retroviral vector (GALV pseudotyped) for an international X-linked SCID trial with average unconcentrated titers of 106 IU/mL in all viral harvests. In summary, the process developed at the Cincinnati Children’s Hospital Vector Production Facility allows for large scale closed-system production of high-titer retroviral vectors for clinical trials using transient transfection.

Figure Figure
Coating of AAV Vectors with Human Albumin Blocks Antibody Binding and Enables Transduction of Human Hepatocytes in the Presence of Neutralizing Antibodies
Authors: Wright J. Fraser
Published: October 2019

Neutralization of recombinant adeno-associated virus (AAV) gene transfer vectors by pre-existing antibodies is a significant barrier to clinical gene transfer using systemic administration. In a recent clinical trial for hemophilia B, while efficient gene transfer and therapeutic levels of hFIX were achieved in a human subject with low pre-existing antibodies to AAV2, no gene transfer was observed in a subject with a modest pre-existing AAV2 antibody titer of 1:17 who received the same dose (Manno et al. Nature Med.2006;12:342–347). With the objective to achieve consistent and efficient transduction in the presence of AAV antibodies, ‘humanized’ AAV vectors were generated by covalent attachment of human albumin (HSA) to the surface of purified AAV2 by cross linking the single free thiol (Cys34) of HSA to lysine residues on the vector surface using suflosuccinimidyl 4 [N-maleimidomethyl] cyclohexane-1-caboxylate (Sulfo-SMCC). Variable levels of HSA substitution up to 65 HSA molecules per virion were achieved, corresponding to a maximum of approximately one HSA molecule per virion capsid protein. Analysis by SDS-PAGE of the modified vector revealed the presence of a 130kDA polypeptide species in the conjugated vector, not observed in the unmodified vector, that was consistent with predicted molecular weight obtained by covalent attachment of HSA with AAV capsid protein VP3. Dynamic light scattering demonstrated an average radius of 16.6 nm for the HSA-conjugated vector compared with 11.6 nm for unconjugated vector, consistent with coating of the vector surface with HSA. Analysis by flow cytometry demonstrated that the binding of AAV2-specific monoclonal antibody A20 to unmodified vectors was progressively reduced to background levels in HSA-conjugated vectors with increasing HSA substitution. Quantitative analysis by Biacore indicated that the association rate and available sites per virion for antibody binding were reduced > 10- fold in HSA-conjugated vectors. The conjugated vectors were significantly more resistant than unmodified vectors to neutralization by AAV antibody, demonstrating comparable transduction in the presence of 16-fold higher concentration of MAb A20 following transduction of cultured human hepatocytes (HepG2). In conclusion, covalent coupling of HSA to the surface of recombinant AAV2 is demonstrated as a feasible method to modify AAV vectors to increase their resistance to neutralizing antibodies. ‘Humanized’ AAV vectors such as HSA-coated AAV that effectively mask viral epitopes recognized by antibodies and display human epitopes may enable more consistent transduction following systemic administration in vivo, and enable vector readministration.