Paul C. Hendrie

Clofarabine with high dose cytarabine and granulocyte colony-stimulating factor (G-CSF) priming for relapsed and refractory acute myeloid leukaemia

This phase I/II study was conducted to determine the maximum tolerated dose, toxicity, and efficacy of clofarabine in combination with high dose cytarabine and granulocyte colony‐stimulating factor (G‐CSF) priming (GCLAC), in the treatment of patients with relapsed or refractory acute myeloid leukaemia (AML). Dose escalation of clofarabine occurred without dose‐limiting toxicity, so most patients were treated at the maximum dose, 25 mg/m2 per day with cytarabine 2 g/m2 per day, each for 5 d, and G‐CSF 5 μg/kg, beginning the day before chemotherapy and continuing daily until neutrophil recovery. The complete remission (CR) rate among the 46 evaluable patients was 46% (95% confidence interval [CI] 31–61%) and the CR + CR but with a platelet count <100 × 109/l rate was 61% (95% CI 45–75%). Multivariate analysis showed that responses to GCLAC were independent of age, cytogenetic risk category, and number of prior salvage regimens. GCLAC is highly active in relapsed and refractory AML and warrants prospective comparison to other regimens, as well as study in untreated patients.

A Rapid and Quantitative Assay for Measuring Neighboring Gene Activation by Vector Proviruses

A simple, quantitative assay for measuring the oncogenic potential of integrating vectors is needed in order to improve vector design and safety. In this study, we have developed a transient plasmid-based assay to measure the activation of a reporter gene by an adjacent vector provirus. Plasmid pACT contains a luciferase cassette driven by a minimal, enhancerless promoter, into which vector proviruses are inserted upstream for evaluation by luciferase assays and northern blots. In a comparison of analogous vectors based on murine leukemia virus (MLV), human immunodeficiency virus (HIV), and foamy virus (FV), we observed significant enhancer activity and read-through transcription from MLV proviruses, and significant read-through transcription from HIV proviruses. HIV and FV proviruses containing an internal MLV long-terminal repeat (LTR) promoter also had significant enhancer activity, which was not observed with an internal promoter from the murine phosphoglycerate kinase-1 gene, PGK. These results demonstrate that neighboring gene activation can be limited by using internal promoter(s) lacking enhancer activity, especially when present in an FV vector backbone that prevents read-through transcription. Although the pACT assay does not measure oncogenesis directly, it should be useful for screening vectors before more time-consuming and costly animal studies are undertaken.

Chromosomal Integration and Homologous Gene Targeting by Replication-Incompetent Vectors Based on the Autonomous Parvovirus Minute Virus of Mice

ABSTRACT The molecular mechanisms responsible for random integration and gene targeting by recombinant adeno-associated virus (AAV) vectors are largely unknown, and whether vectors derived from autonomous parvoviruses transduce cells by similar pathways has not been investigated. In this report, we constructed vectors based on the autonomous parvovirus minute virus of mice (MVM) that were designed to introduce a neomycin resistance expression cassette (neo) into the X-linked human hypoxanthine phosphoribosyl transferase (HPRT) locus. High-titer, replication-incompetent MVM vector stocks were generated with a two-plasmid transfection system that preserved the wild-type characteristic of packaging only one DNA strand. Vectors with inserts in the forward or reverse orientations packaged noncoding or coding strands, respectively. In human HT-1080 cells, MVM vector random integration frequencies (neo+ colonies) were comparable to those obtained with AAV vectors, and no difference was observed for noncoding and coding strands. HPRT gene-targeting frequencies (HPRT mutant colonies) were lower with MVM vectors, and the noncoding strand frequency was threefold greater than that of the coding strand. Random integration and gene-targeting events were confirmed by Southern blot analysis of G418- and 6-thioguanine (6TG)-resistant clones. In separate experiments, correction of an alkaline phosphatase (AP) gene by gene targeting was nine times more effective with a coding strand vector. The data suggest that single-stranded parvoviral vector genomes are substrates for gene targeting and possibly for random integration as well.

Reduced Genotoxicity of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-term Repopulating Cells Compared to Standard Murine Retrovirus Vectors

Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.

Frequency of allogeneic hematopoietic cell transplantation among patients with high- or intermediate-risk acute myeloid leukemia in first complete remission.

PURPOSE To determine the frequency of allogeneic hematopoietic cell transplantation (HCT) for patients with acute myeloid leukemia (AML) in first complete remission (CR1). PATIENTS AND METHODS Between January 1, 2008, and March 1, 2011, 212 newly diagnosed patients with AML received treatment at our center. Ninety-five patients age less than 75 years with intermediate- or high-risk AML achieved a complete remission, and 21 patients achieved a morphologic remission with incomplete blood count recovery. RESULTS Seventy-eight (67%; 95% CI, 58% to 76%) of 116 patients received HCT at a median of 2.8 months (range, 0.5 to 19 months) from their CR1 date. The median age was 57 years in both the HCT patient group (range, 18 to 75 years) and the non-HCT patient group (range, 24 to 70 years; P = .514). Between the HCT patients and the non-HCT patients, the mean Eastern Cooperative Oncology Group performance status was 1.1 compared with 1.5, respectively (P = .005), and the average HCT comorbidity score within 60 days of CR1 was 1.7 and 2.1, respectively (P = .68). Twenty-nine (76%) of 38 non-HCT patients were HLA typed, and matched donors were found for 13 of these 29 patients (34% of all non-HCT patients). The most common causes for patients not receiving transplantation in CR1 were early relapse (within 6 months) in 12 patients (32%), poor performance status in eight patients (21%), and physician decision in five patients (13%). CONCLUSION HCT can be performed in CR1 in the majority of patients with AML for whom it is currently recommended. The main barriers to HCT were early relapse and poor performance status, highlighting the need for improved therapies for patients with AML of all ages.

Gemtuzumab ozogamicin in combination with vorinostat and azacitidine in older patients with relapsed or refractory acute myeloid leukemia: a phase I/II study

Epigenetic therapeutics such as the histone deacetylase inhibitor, vorinostat, and the DNA methyltransferase I inhibitor, azacitidine, enhance gemtuzumab ozogamicin efficacy in vitro. We therefore investigated vorinostat/azacitidine/gemtuzumab ozogamicin in 52 adults aged 50 years or over with acute myeloid leukemia requiring therapy for first relapse (remission duration ≤12 months) or primary refractory disease in a phase I/II trial. Vorinostat and gemtuzumab ozogamicin were escalated step-wise during the phase I portion of the trial. Vorinostat (400 mg/day orally from Days 1–9), azacitidine (75 mg/m2/day intravenously or subcutaneously from Days 1–7), and gemtuzumab ozogamicin (3 mg/m2/day intravenously on Days 4 and 8) were identified as the maximum tolerated dose. Among the 43 patients treated at this dose, 10 achieved a complete remission and 8 achieved a complete remission with incomplete blood count recovery, for an overall response rate of 41.9% (exact 95% confidence interval (CI): 27.0–57.9%). Four of these 18 patients (2 with complete remission and 2 with complete remission with incomplete blood count recovery) had persistence of minimal residual disease by flow cytometry at the time of best response. Four patients died within 28 days of treatment initiation. Median overall survival for the 18 patients achieving complete remission/complete remission with incomplete blood count recovery was significantly longer than for those 21 patients who failed therapy but lived at least 29 days after treatment initiation (224.5 days (range 70–798) vs. 95 days (range 36–900); P=0.0023). These data indicate that vorinostat/azacitidine/gemtuzumab ozogamicin has activity in this difficult-to-treat acute myeloid leukemia patient subset. (ClinicalTrials.gov: identifier 00895934).

Phase II study of tosedostat with cytarabine or decitabine in newly diagnosed older patients with acute myeloid leukaemia or high-risk MDS

Tosedostat, an oral aminopeptidase inhibitor, has synergy with cytarabine and hypomethylating agents. We performed a Phase II trial to determine rates of complete remission (CR) and survival using tosedostat with cytarabine or decitabine in older patients with untreated acute myeloid leukaemia (AML) or high‐risk myelodysplastic syndrome (MDS). Thirty‐four patients ≥60 years old (median age 70 years; range, 60–83) were randomized to receive tosedostat (120 mg on days 1–21 or 180 mg continuously) with 5 d of either cytarabine (1 g/m2/d) or decitabine (20 mg/m2/d) every 35 d. Twenty‐nine patients (85%) had AML, including 15 (44%) with secondary AML/MDS, and 5 (15%) had MDS‐refractory anaemia with excess blasts type 2. The CR/CR with incomplete count recovery (CRi) rate was 53% [9 in each arm; 14 CR (41%) and 4 CRi (12%)], attained in 6 of 14 patients with adverse cytogenetics and 4 of 7 with FLT3‐internal tandem duplication mutations. Median follow‐up was 11·2 months (range, 0·5–22·3), and median survival was 11·5 months (95% confidence interval, 5·2–16·7). Twenty‐three patients (67·6%) were treated as outpatients and 10 of these patients required hospitalization for febrile neutropenia. No Grade 3–4 non‐haematological toxicities required withdrawal from study. Tosedostat with cytarabine or decitabine is tolerated in older patients with untreated AML/MDS, results in a CR/CRi rate of >50%, and warrants further study in larger trials.

Value of routine ‘day 14’ marrow exam in newly diagnosed AML

2013). 8 Vainstein V, Buckley SA, Shukron O, Estey EH, Abkowitz JL, Wood BL et al. Rapid rate of peripheral blood blast clearance accurately predicts complete remission in acute myeloid leukemia. Leukemia 2014; 28: 713–716. Differential association of calreticulin type 1 and type 2 mutations with myelofibrosis and essential thrombocytemia: relevance for disease evolution Leukemia (2015) 29, 249–252; doi:10.1038/leu.2014.270 Recent advances in myeloproliferative neoplasms (MPNs) have highlighted the prevalence of mutations in the calreticulin gene (CALR), bringing a major new actor in these disorders. Numerous studies have highly contributed to understanding of the role of JAK2 and MPL in the pathogenesis of MPN development and progression. In contrast to JAK2 mutations that are associated in a causative manner with polycythemia vera, myelofibrosis (MF) or essential thrombocytemia (ET), CALR mutations are only associated with ET and MF. Indeed, CALR mutations were reported in 25% of ET and in 35% of MF patients who were non-mutated for JAK2 and MPL. All recurrent CALR mutations that have been observed lead to a frame-shift generating a common 36 amino-acid C-terminal end and loss of the KDEL motif. Among these mutations, two variants are largely predominant: type 1, a 52-bp deletion (p.L367fs*46) and type 2, a 5-bp insertion (p.K385fs*47). They account for 85% of the CALR mutations in ET and primary myelofibrosis (PMF). There is strong evidence that the clinical features of CALR and JAK2-mutated ET and PMF are different and represent two facets of these disorders, although they both activate the JAK/STAT signaling pathway. It remains possible that the different CALR mutants exert different signaling activities. Thus, it would be important to characterize the potential differences between the different CALR mutations and also the phenotype of MPN devoid of JAK2, MPL and CALR mutations, now called triple-negative MPNs to understand whether they represent a homogeneous population. A total of 572 MPN patients negative for JAK2 and MPL mutations were collected according to sex, age at diagnosis and main clinical characteristics from several French and Belgian hospitals under the auspices of French Intergroup of Myeloproliferative disorders. The mutational status of CALR was determined using previously described high-resolution sizing of fluorescent dye-labeled PCR amplification of exon 9, with Sanger sequencing controls. Beyond its contribution to mutant detection, highresolution sizing also allows to estimate the allelic burden. Laboratory data and clinical parameters were obtained at diagnosis. The GraphPad Prism statistical package (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform statistical analysis on these data, as described in Supplementary Information. The median age at diagnosis of the entire patient series was 59 years (range, 7–92); 241 were males (43.1%). In our series, 396 patients were diagnosed as ET (including potential unidentified prefibrotic stage of myelofibrosis), 108 as MF (including PMF and post-ET MF) and 68 as mixed myelodysplastic syndrome (MDS)/ MPN. We identified mutations localized in exon 9 of CALR in 368 patients (63.3%). The remaining 204 patients were designated as triple negative. The global distribution of CALR mutations in our series was similar to previous studies with 206 type 1 mutations (56%), 119 type 2 mutations (32%) and 43 other mutations (12%) representing 17 different CALR variants (Figure 1a). Importantly, nine were previously described but eight represented new variants (Figure 1b). Notably one of them, p.Glu378Cysfs*36, shortened the consensus C-terminal end to 33 amino acids only. In contrast to the rather low numbers of JAK2 and MPL mutation variants, the growing number of CALR genomic variants identified implies that these mutants might not be completely biologically equivalent, despite the common loss of the KDEL motif and the acquisition of a new functional domain. Differences in the sequence of the C-terminal domain could indeed reach up to 25 amino acids, which is predicted to change the properties of the protein. Our striking result is the distribution of CALR mutation type according to the MPN type. In MF there is an overrepresentation of type 1 CALR mutation (70%) and an underrepresentation of type 2 CALR mutation (13%) as compared with patients with ET that have a distribution of CALR mutations comparable to the global series of patients (Figure 1a; P= 0.0002). In MPNs, it has been highlighted that different allelic burdens of JAK2V617F and MPLW515K/L are associated with different phenotypes. In our series, a higher allelic burden of CALR mutation was found in MF (Po0.0001; Table 1). Even if it affects a small number of patients, it is to be noted that a high allelic burden (over 60%) was more frequent in MF (5/35 = 14.3%) than in ET (6/158 = 3.8%; P= 0.0256). This highly suggests the presence of rare homozygous mutations as described by Klampfl. However, and contrary to that study, we observed a high allelic burden mainly Accepted article preview online 12 September 2014; advance online publication, 14 October 2014 Letters to the Editor

Resource Utilization and Safety of Outpatient Management Following Intensive Induction or Salvage Chemotherapy for Acute Myeloid Leukemia or Myelodysplastic Syndrome: A Nonrandomized Clinical Comparative Analysis

IMPORTANCE Adults with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) typically remain hospitalized after induction or salvage chemotherapy until blood cell count recovery, with resulting prolonged inpatient stays being a primary driver of health care costs. Pilot studies suggest that outpatient management following chemotherapy might be safe and could reduce costs for these patients. OBJECTIVE To compare safety, resource utilization, infections, and costs between adults discharged early following AML or MDS induction or salvage chemotherapy and inpatient controls. DESIGN Nonrandomized, phase 2, single-center study conducted at the University of Washington Medical Center. Over a 43-month period (January 1, 2011, through July 31, 2014), 178 adults receiving intensive AML or MDS chemotherapy were enrolled. After completion of chemotherapy, 107 patients met predesignated medical and logistical criteria for early discharge, while 29 met medical criteria only and served as inpatient controls. INTERVENTIONS Early-discharge patients were released from the hospital at the completion of chemotherapy, and supportive care was provided in the outpatient setting until blood cell count recovery (median, 21 days; range, 2-45 days). Controls received inpatient supportive care (median, 16 days; range, 3-42 days). MAIN OUTCOMES AND MEASURES We analyzed differences in early mortality, resource utilization including intensive care unit (ICU) days, transfusions per study day, and use of intravenous (IV) antibiotics per study day), numbers of infections, and total and inpatient charges per study day among early-discharge patients vs controls. RESULTS Four of the 107 early-discharge patients and none of the 29 control patients died within 30 days of enrollment (P=.58). Nine early-discharge patients (8%) but no controls required ICU-level care (P=.20). No differences were noted in the median daily number of transfused red blood cell units (0.27 vs 0.29; P=.55) or number of transfused platelet units (0.26 vs 0.29; P=.31). Early-discharge patients had more positive blood cultures (37 [35%] vs 4 [14%]; P=.04) but required fewer IV antibiotic days per study day (0.48 vs 0.71; P=.01). Overall, daily charges among early-discharge patients were significantly lower than for inpatients (median,

Transfusion-associated graft-versus-host disease in a liver transplant recipient: an unusual presentation and review of the literature

3840 vs