The Impact of Aging of Hematopoietic Stem and Progenitor Cells (HSPCs) in Non-Human Primates As Interrogated By Genetic Barcode Clonal Tracking

Abstract Aging of the hematopoietic system is associated with a number of observations, including diminished regenerative potential, skewed lineage differentiation, increased incidence of anemia, and higher rates of neoplastic transformation. Despite advanced age being a strong poor prognostic factor, an increasing number of older patients are receiving hematopoietic stem and progenitor cell (HSPC) transplantation. Most previous investigations of the effects of aging on hematopoiesis have been obtained in murine models. The rhesus macaque is a powerful model to study human hematopoiesis and aging, based on a close phylogenetic relationship to humans, and similar telomere lengths, lifespans and aging phenotypes. To quantitatively elucidate the age-related changes that compromise hematopoietic function at a clonal level, we applied a genetic barcoding approach to quantitatively track the clonal behavior of HSPCs in young versus old macaques following autologous transplantation. We delivered high diversity barcodes via lentiviral transduction of CD34+ HSPC (detailed in our prior study: Wu et al Cell Stem Cell, 2014), allowing quantitative tracking of the output of thousands of individual HSPC clones labeled by unique barcodes, over time and in various lineages post-transplantation. We successfully transplanted barcoded HSPCs into 2 macaques aged 19 and 25 years, constituting "old" macaques based on an average lifespan in captivity of 20-30 years, and compared results to clonal patterns observed in 5 "young" macaques aged 3-5 years. Both old macaques engrafted promptly, and CD3+ T cells, CD3-CD20+ B cells, CD33+ Granulocytes (Gr), CD3-CD20-CD14+ Monocytes (Mo), and CD3-CD20-CD14-CD16+/or CD56+ NK cells were purified from the peripheral blood monthly following transplantation. In terms of overall polyclonality and diversity (Shannon index), analyzed through 4 months to date, there were no marked differences between the old and young recipients, with thousands of individual clones contributing to hematopoiesis in both sets of animals during the initial post-transplant time period studied. However, there were marked differences in the patterns of clonal lineage relationships between young and old animals, as assessed via pairwise Pearson correlations of all contributing clones as well as clustering algorithms allowing interrogation of patterns of clonal behavior. In both young and old, there was no correlation (i.e. no shared clones and thus no shared progenitors) between lineages at 1m, and clones contributing at 1m did not contribute to any lineage at 3m or later, indicating the existence of short-term, lineage-restricted progenitors in both age groups. By 3m in young animals, B and Gr/Mo became correlated, and by 3-6m, B/T/Mo/Gr multilineage clones appeared and constituted the majority of hematopoietic output. However, in old animals clones contributing to Gr/Mo versus B or T lineages remained almost completely distinct or markedly biased, up to 4m studied to date, without evidence for multi-lineage clones (Fig 1). In both young and old animals, the NK lineage remained clonally distinct, as previously reported for young animals. In summary, we transplanted 2 aged macaques with barcoded CD34+ HSPCs, and discovered a pattern of clonal reconstitution distinct from that in young animals, with persistent unilineage or highly-biased myeloid and B lymphoid progenitors in the aged animals. Longer follow-up will be required to determine if this biased pattern persists, and will be presented. This approach should improve our understanding of disorders of hematopoiesis in the elderly, and help improve transplantation and other therapies in this vulnerable patient population. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Impact of Cytopenia Following Unmanipulated Haploidentical Hematopoietic Stem Cell Transplantation Using Post-Transplant Cyclophoshamide

Blood ◽

10.1182/blood.v128.22.2281.2281 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2281-2281

Author(s):

Villetard Ferdinand ◽

Stefania Bramanti ◽

Samia Harbi ◽

Sabine Fürst ◽

Catherine Faucher ◽

...

Keyword(s):

Bone Marrow ◽

Cell Transplantation ◽

Disease Risk ◽

Conflicts Of Interest ◽

Risk Index ◽

Conditioning Regimen ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Cell Dose ◽

The Impact

Abstract Introduction Allogeneic transplantation from a haploidentical donor (HaploSCT) is an alternative strategy in the treatment of hematologic malignancies in absence of HLA-identical donor. Recent studies reported similar outcome after HaploSCT compared to HLA-identical transplantation in different settings (Bashey, JCO 2013; Wang, Blood 2015; Gosh, JCO 2016). Although survivals seemed promising after HaploSCT, hematopoietic recovery following such a mismatched transplantation could represent a limitation. Thus, our series aims to evaluate hematological recovery after HaploSCT using a post transplantation cyclophosphamide (PT-Cy) platform. Methods This retrospective monocentric study included consecutive patients with following criteria: adults with hematological malignancies; bone marrow or peripheral blood T-replete HaploSCT from 2011 to 2015; non-myeloablative (Baltimore approach) or reduced intensity conditioning (busulfan-based) regimen; PT-Cy as part of GVHD prophylaxis. Patients with primary graft failure were excluded. Absolute neutrophil count (ANC), red cells (RCT) or platelet transfusion (PT) requirements on day 30 (D30) and day 100 (D100) were analyzed among disease-free patients. We first separately evaluated the rate of patients with significant cytopenia in each lineage (defined by ANC < 1 G/L, RCT need, PT need) and searched for impact of pre-transplantation factors on cytopenia (multivariate analyses by binary logistic regression). Then, we evaluated outcome by D30- and D100-landmark analyses according to cytopenia. Results One hundred and forty six patients with a median age of 56 years (range: 19-73) were analyzed: 142 and 117 were evaluable at D30 (4 early deaths) and D100 (17 deaths, 11 relapses), respectively. At D30, 20% of patients had ANC<1G/L, 67% needed RCT and 63% needed PT. Corresponding values at D100 were 20%, 42% and 28%, respectively (Figure 1). At D30: the use of PBSC (HR 9.5, p=0.002) was significantly associated with ANC>1G/L at D30; the use of NMAC Baltimore schema (HR 0.3, p=0.012) and CD34+ cell dose > median (HR 0.4, p=0.041) decreased PT needs while hematopoietic cell transplantation comorbidity index (HCT-CI)≥3 (HR 3.3, p=0.004) was associated with PT needs; no factor was found to significantly influence RCT. At D100: Age>60 years (HR 2.4, p=0.045), female to male HaploSCT (HR 3.3, p=0.020) and HCT-CI≥3 (HR 3.7, p=0.006) were significantly associated with higher risk of RCT need; female to male HaploSCT (HR 3.6, p=0.015) and HCT-CI≥3 (HR 6.9, p=0.001) were associated with PT needs; no factor was found to significantly influence ANC. With a median follow up of 25 months (range: 5-55), cox multivariate model with adjustment by age (continuous), disease risk index (low/intermediate vs high/very high), HCT-CI (0-2 vs ≥3), conditioning regimen (baltimore vs. busulfan-based) and graft source (bone marrow vs PBSC) showed that ANC<1 G/L was strongly associated with higher NRM (HR 2.9, p=0.011) and shorter OS (HR 3.4, p<0.001), overcoming the impact of RCT and PT needs (Figure 2A and 2B). In contrast, D100 analysis showed that PT need was the most determinant factor of increased NRM (HR 13.7, p=0.013) and poor OS (HR 7.3, p=0.003), while both D100 ANC and RCT needs did not impact outcome (Figure 2C and 2D). Discussion We found that cytopenia remain a concern after HaploSCT, leading to increased NRM and OS. The absence of ANC>1G/L at D30 as well as the need of PT at D100 may be considered as a strong post transplantation factor predicting poor outcome. Some pre-transplantation factors of cytopenia have been identified, such as CD34+ cell dose, sex mismatch and graft source. Among them, some may help for donor selection while the optimal donor for HaploSCT is still unknown. Moreover, better neutrophil recovery at D30 is achieved with the use of PBSC. CD34+ optimal cell dose in this setting remains also to be determined. In addition, post transplantation events such GVHD and/or infections should be evaluate to explore their interactions with such cytopenia, aiming to develop early therapeutic interventions. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Allogeneic stem cell transplantation may overcome the adverse impact of myelofibrosis on the prognosis of myelodysplastic syndrome

Experimental Hematology and Oncology ◽

10.1186/s40164-021-00238-x ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Xiangzong Zeng ◽

Li Xuan ◽

Zhiping Fan ◽

Yu Zhang ◽

Ke Zhao ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Disease Free Survival ◽

Adverse Impact ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Poor Prognostic Factor ◽

Cox Analysis ◽

The Impact

Abstract Purpose Myelofibrosis (MF) may serve as a poor prognostic factor in myelodysplastic syndromes (MDS). This study explored the impact of allogeneic hematopoietic stem cell transplantation (allo-HSCT) on the outcome of MDS patients with MF. Patients and Methods Three hundred and sixteen MDS patients were enrolled in this retrospective study. Based on the degree of MF, we divided the patients into 2 groups: grade 0–1 (MF-0/1) and grade 2–3 (MF-2/3) groups. The clinical features, treatments, and prognosis in MDS patients with MF were analyzed. Results Forty-three (13.6%) patients were diagnosed as MF-2/3. Complex karyotypes were more common in the MF-2/3 compared to MF-0/1 groups (P = 0.002). The overall response rate (ORR) of cytoreduction was 49.0%, along with 53.3% in the MF-0/1 and 16.7% in MF-2/3 groups (P = 0.017). In total, 141 patients underwent allo-HSCT, including 121 in the MF-0/1 and 20 in MF-2/3 groups. The median time to neutrophil reconstruction was 12 (range: 7–34) and 14 (range: 10–45) days (P = 0.005), and platelet reconstruction was 14 (range: 8–68) and 18 (range: 8–65) days (P = 0.045) in the MF-0/1 and MF-2/3 groups, respectively. However, the cumulative incidence of neutrophil and platelet engraftment achieved at day + 30 was not different between the two groups (P = 0.107, P = 0.303, respectively). Non-relapse mortality, relapse, and acute and chronic graft-versus-host disease were similar between the two groups (all P > 0.05). Among patients with allo-HSCT, the 2-year overall survival (OS) was 68.5% (95% CI: 60.1–76.9%) and 68.4% (95% CI: 47.4–89.4%) in the MF-0/1 and MF-2/3 groups, respectively, (P = 0.636). Among patients without allo-HSCT, the 2-year OS was 49.9% (95% CI: 40.7–59.1%) and 19.2% (95% CI: 0–39.6%) in the MF-0/1 and MF-2/3 groups, respectively, (P = 0.002). In multivariate cox analysis, complex karyotype was an unfavorable factor for relapse (HR, 4.16; P = 0.006), disease-free survival (DFS) (HR, 2.16; P = 0.020), and OS (HR, 2.47; P = 0.009) post-transplantation. Conclusion Patients with MF-2/3 have more complex karyotypes and lower ORR of cytoreduction in MDS. Among patients without allo-HSCT, patients with MF-2/3 have a worse prognosis than those with MF-0/1. However, the adverse impact of MF on prognosis may be overcome by allo-HSCT.

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Human Genetic Diversity Alters Therapeutic Gene Editing Off-Target Outcomes

Blood ◽

10.1182/blood-2021-152429 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3993-3993

Author(s):

Linda Yingqi Lin ◽

Samuele Cancellieri ◽

Jing Zeng ◽

Francesco Masillo ◽

My Anh Nguyen ◽

...

Keyword(s):

Genetic Diversity ◽

Clinical Trials ◽

Genome Editing ◽

Gene Editing ◽

Conflicts Of Interest ◽

African Ancestry ◽

Great Promise ◽

Personal Genome ◽

Hematopoietic Stem ◽

The Impact

Abstract CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to predict and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia (Frangoul et al. NEJM 2021). We find that the top predicted off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF) = 4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (~9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. This report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We expect that variant-aware off-target assessment will be required for therapeutic genome editing efforts going forward, including both ongoing and future clinical trials, and we provide a powerful approach for comprehensive off-target prediction. CRISPRme is available at crisprme.di.univr.it. Disclosures No relevant conflicts of interest to declare.

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Molecular pathogenesis of progression to myeloid leukemia from TET-insufficient status

Blood Advances ◽

10.1182/bloodadvances.2019001324 ◽

2020 ◽

Vol 4 (5) ◽

pp. 845-854 ◽

Cited By ~ 3

Author(s):

Raksha Shrestha ◽

Mamiko Sakata-Yanagimoto ◽

Koichiro Maie ◽

Motohiko Oshima ◽

Masatomo Ishihara ◽

...

Keyword(s):

Median Survival ◽

Myeloid Leukemia ◽

Recurrent Events ◽

Loss Of Function ◽

Genetic Event ◽

Hematopoietic Stem ◽

Age Related ◽

Whole Exome ◽

Recurrent Mutations ◽

The Impact

Abstract Loss-of-function mutations in ten-eleven translocation-2 (TET2) are recurrent events in acute myeloid leukemia (AML) as well as in preleukemic hematopoietic stem cells (HSCs) of age-related clonal hematopoiesis. TET3 mutations are infrequent in AML, but the level of TET3 expression in HSCs has been found to decline with age. We examined the impact of gradual decrease of TET function in AML development by generating mice with Tet deficiency at various degrees. Tet2f/f and Tet3f/f mice were crossed with mice expressing Mx1-Cre to generate Tet2f/wtTet3f/fMx-Cre+ (T2ΔT3), Tet2f/fTet3f/wtMx-Cre+ (ΔT2T3), and Tet2f/fTet3f/fMx-Cre+ (ΔT2ΔT3) mice. All ΔT2ΔT3 mice died of aggressive AML at a median survival of 10.7 weeks. By comparison, T2ΔT3 and ΔT2T3 mice developed AML at longer latencies, with a median survival of ∼27 weeks. Remarkably, all 9 T2ΔT3 and 8 ΔT2T3 mice with AML showed inactivation of the remaining nontargeted Tet2 or Tet3 allele, respectively, owing to exonic loss in either gene or stop-gain mutations in Tet3. Recurrent mutations other than Tet3 were not noted in any mice by whole-exome sequencing. Spontaneous inactivation of residual Tet2 or Tet3 alleles is a recurrent genetic event during the development of AML with Tet insufficiency.

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Allogeneic Hematopoietic Stem Cell Transplantation Cures CLL: A Retrospective Analysis from the SFGM-TC Registry.

Blood ◽

10.1182/blood.v108.11.2991.2991 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2991-2991

Author(s):

Mauricette Michallet ◽

Quoc-Hung Lê ◽

Jean-Paul Vernant ◽

Franck E. Nicolini ◽

Jean-Luc Harousseau ◽

...

Keyword(s):

Autologous Transplantation ◽

Disease Status ◽

Allogeneic Transplantation ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Cord Blood Cell ◽

The Status ◽

The Impact ◽

Long Term Survivors

Abstract This retrospective study concerned 471 B-CLL patients registered in the SFGM-TC registry from Apr 1,984 to Feb 2,005, who underwent either autologous transplantation (n=313, 138 F and 175 M, median age = 54, 236 PBSC and 77 BM) or allogeneic transplantation (n=158, 78 F and 80 M, median age = 49, 76 PBSC, 81 BM and 1 cord blood cell transplant from 17 related and 141 unrelated donors). Among alloT patients, 50 were ABO incompatible and 70 sex-mismatched. The median interval diagnosis-transplantation was 32 months for autoT and 51 months for alloT. Just before conditioning 302 autoT and 143 alloT were evaluated for the disease status: 100 and 26 patients were in CR, 170 and 55 were in PR, 4 and 13 in stable disease (SD), 28 and 49 in progressive disease (PD) for autoT and alloT respectively. Among alloT patients, 73 received reduced intensity conditioning (RIC) and 85 standard conditioning (72 Cyt+TBI, 33 Fluda+TBI, 23 Fluda+Bu+ATG, 8 Cyt+Bu and 21 other). Before autoT the conditioning consisted of 224 Cyt+TBI, 45 BEAM and 44 other. After alloT, 71 patients developed an aGVHD ≥ grade II and 60 developed a cGVHD (25 limited and 35 extensive). The non-relapse mortality at 1 year was 29%. With a mean follow-up of 28 months for autoT and 40 months for alloT, the probabilities of 3-year, 5-year and 8-year overall survival were 80%, 66%, 45.5% after autoT and 52%, 48% and 35% after alloT respectively. An analysis aimed to determine the percentage of long-term survivors, or patients censored on the final plateau of survival curves was performed on alloT and autoT groups. A mixture model, gfcure with Splus statistical package determined the percentages of long-term survivors and its adequacy was verified graphically. The percentage of long-term survivors for the autoT group was 1.2%, with a mean survival length for uncured population of 160 months. Fig A shows that both curves were close and consequently shows good adequacy and the absence of a final plateau. The percentage of long-term survivors for alloT was 34.03% (figure1). Fig B shows rather good adequacy. The study of the impact of usual prognosis factors (age, time diagnosis-transplant, sex match, HLA match, CMV status, type of conditioning, BM or PBSC, ABO compatibility and disease status before transplantation) on the percentage of long-term survivors showed that only the status of disease at transplant had a significant impact: (CR vs SD or PD, HR: 0.11 [0.02–0.5] p=0.01 and PR vs SD or PD, HR: 0.30 [0.09–0.96] p=0.04). This study pointed out the possibility of curing B-CLL patients who responded to conventional chemotherapy with allogeneic transplantation rather than with autologous transplantation. Figure Figure Figure Figure

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Re-Exposure of Cord Blood (CB) to Non-Inherited Maternal HLA Antigens (NIMA) Improves Transplant Outcome in Patients (pts) with Hematologic Malignancies and May Reduce Relapse.

Blood ◽

10.1182/blood.v114.22.661.661 ◽

2009 ◽

Vol 114 (22) ◽

pp. 661-661

Author(s):

Jon J van Rood ◽

Cladd E Stevens ◽

Jacqueline Smits ◽

Carmelita Carrier ◽

Carol Carpenter ◽

...

Keyword(s):

Conflicts Of Interest ◽

Chronic Gvhd ◽

Hla Antigens ◽

Human Leukocyte ◽

Hematologic Malignancies ◽

Hematopoietic Stem ◽

Leukocyte Antigens ◽

Grade Ii ◽

The Impact ◽

Related Mortality

Abstract Abstract 661 CB hematopoietic stem cell transplantation (CBT) can be successful even if donor and recipient are not fully matched for human leukocyte antigens (HLA). This may result, at least in part, from tolerance-inducing events during pregnancy, but this concept has not been tested to date. Hence we analyzed the impact of fetal exposure to NIMA of the HLA-A, -B antigens or -DRB1 alleles on the outcome of 1121 pts with hematologic malignancies. All pts received single CB units provided by the NYBC, for treatment of ALL (N=451), AML (N=376), CML (N=116), MDS (N=79), other (N=99); 22% were transplanted in advanced stage. Median age was 9.7 years (range: 0.1-67); 29% of recipients were >16 years. Most pts (96%) received myeloablative cytoreduction. Sixty-two pts received fully matched grafts while 1059 received units mismatched (MM) for one or two HLA antigens. Of these, 79 (7%) had a MM antigen which was identical to a donor NIMA (Example: Pt: A1, A3; CBU: A1, A2; mother-CBU: A1, A3; A3 is NIMA). NIMA match was found in 25 recipients with one HLA MM and 54 of those with two MM. The NIMA match was identified after the transplant and was not used in unit selection. In multivariate analyses, NIMA matched transplants (NMTs), showed faster neutrophil recovery (RR=1.3, p=0.043), even for grafts with cell dose <3×107 (RR=1.6, p=0.053). There was no difference in the incidence of acute (grade II-IV) or chronic GvHD. 3-year relapse risk (cumulative incidence 22%) was reduced compared to 1 or 2 HLA MM no NIMA matched transplants, especially in pts with myelogenous malignancies given units with 1 HLA MM (RR=0.2, p=0.074). Further, 3-year transplant-related mortality was reduced (RR=0.7, p=0.034), particularly in pts ≥5 years old (RR=0.5, p=0.006), as was the 3-year overall mortality (RR= 0.7, p=0.029 and RR=0.6, p=0.015, respectively). As a result, in the NMTs, treatment failure (relapse or death) was significantly lower, particularly in pts ≥5 years (RR=0.7, p=0.019) and DFS was significantly improved (figure) and was similar to that of the 0 HLA MM group. These findings are the first indication that donor exposure to NIMA can improve post-transplant survival in unrelated CBT and might reduce relapse. We propose to include the NIMA of CB units in search algorithms. Thus, for pts lacking fully HLA matched grafts, HLA MM but NIMA matched CB units could be selected preferentially, since no adverse effects were seen. This strategy of selecting HLA MM grafts with optimal outcome effectively “expands” the current CB Inventory several-fold.Patient GroupNRR(95% Cl)p value0 MM360.5(0.3–0.8)0.0051 MM / NIMA Match180.4(0.2–0.9)0.0262 MM / NIMA Match400.8(0.5–1.2)0.3091 MM / No NIMA Match229reference group2 MM / No NIMA Match4871.1(0.9–1.3)0.365 Disclosures: No relevant conflicts of interest to declare.

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A Modified HIV1-Based Lentiviral Vector Can Transduce Rhesus Hematopoietic Repopulating Cells as Efficiently as An SIV Vector in An Autologous Transplantation Model.

Blood ◽

10.1182/blood.v114.22.692.692 ◽

2009 ◽

Vol 114 (22) ◽

pp. 692-692

Author(s):

Naoya Uchida ◽

Phillip W Hargrove ◽

Kareem Washington ◽

Coen J. Lap ◽

Matthew M. Hsieh ◽

...

Keyword(s):

T Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Autologous Transplantation ◽

Vector System ◽

Transduction Efficiency ◽

Large Animal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 692 HIV1-based vectors transduce rhesus hematopoietic stem cells poorly due to a species specific block by restriction factors, such as TRIM5αa which target HIV1 capsid proteins. The use of simian immunodeficiency virus (SIV)-based vectors can circumvent this restriction, yet use of this system precludes the ability to directly evaluate HIV1-based lentiviral vectors prior to their use in human clinical trials. To address this issue, we previously developed a chimeric HIV1 vector (χHIV vector) system wherein the HIV1-based lentiviral vector genome is packaged in the context of SIV capsid sequences. We found that this allowed χHIV vector particles to escape the intracellular defense mechanisms operative in rhesus hematopoietic cells as judged by the efficient transduction of both rhesus and human CD34+ cells. Following transplantation of rhesus animals with autologous cell transduced with the χHIV vector, high levels of marking were observed in peripheral blood cells (J Virol. 2009 Jul. in press). To evaluate whether χHIV vectors could transduce rhesus blood cells as efficiently as SIV vectors, we performed a competitive repopulation assay in two rhesus macaques for which half of the CD34+ cells were transduced with the standard SIV vector and the other half with the χHIV vector both at a MOI=50 and under identical transduction conditions. The transduction efficiency for rhesus CD34+ cells before transplantation with the χHIV vector showed lower transduction rates in vitro compared to those of the SIV vector (first rhesus: 41.9±0.83% vs. 71.2±0.46%, p<0.01, second rhesus: 65.0±0.51% vs. 77.0±0.18%, p<0.01, respectively). Following transplantation and reconstitution, however, the χHIV vector showed modestly higher gene marking levels in granulocytes (first rhesus: 12.4% vs. 6.1%, second rhesus: 36.1% vs. 27.2%) and equivalent marking levels in lymphocytes, red blood cells (RBC), and platelets, compared to the SIV vector at one month (Figure). Three to four months after transplantation in the first animal, in vivo marking levels plateaued, and the χHIV achieved 2-3 fold higher marking levels when compared to the SIV vector, in granulocytes (6.9% vs. 2.8%) and RBCs (3.3% vs. 0.9%), and equivalent marking levels in lymphocytes (7.1% vs. 5.1%) and platelets (2.8% vs. 2.5)(Figure). Using cell type specific surface marker analysis, the χHIV vector showed 2-7 fold higher marking levels in CD33+ cells (granulocytes: 5.4% vs. 2.7%), CD56+ cells (NK cells: 6.5% vs. 3.2%), CD71+ cells (reticulocyte: 4.5% vs. 0.6%), and RBC+ cells (3.6% vs. 0.9%), and equivalent marking levels in CD3+ cells (T cells: 4.4% vs. 3.3%), CD4+ cells (T cells: 3.9% vs. 4.6%), CD8+ cells (T cells: 4.2% vs. 3.9%), CD20+ cells (B cells: 7.6% vs. 4.8%), and CD41a+ cells (platelets: 3.5% vs. 2.2%) 4 months after transplantation. The second animal showed a similar pattern with higher overall levels (granulocytes: 32.8% vs. 19.1%, lymphocytes: 24.4% vs. 17.6%, RBCs 13.1% vs. 6.8%, and platelets: 14.8% vs. 16.9%) 2 months after transplantation. These data demonstrate that our χHIV vector can efficiently transduce rhesus long-term progenitors at levels comparable to SIV-based vectors. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in the large animal model, especially for granulocytic or RBC diseases. Disclosures: No relevant conflicts of interest to declare.

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Autologous Hematopoietic Stem Cell Transplantation (autoHSCT) in CLL: First Results of An EBMT Randomized Trial Comparing Autotransplant Versus Wait and Watch.

Blood ◽

10.1182/blood.v114.22.877.877 ◽

2009 ◽

Vol 114 (22) ◽

pp. 877-877

Author(s):

Mauricette Michallet ◽

Peter Dreger ◽

Laurent Sutton ◽

Ronald Brand ◽

Sue Richards ◽

...

Keyword(s):

Conflicts Of Interest ◽

Progression Free Survival ◽

Disease Status ◽

Primary Objective ◽

Phase Iii ◽

Induction Treatment ◽

Hematopoietic Stem ◽

Binet Stage ◽

The Impact

Abstract Abstract 877 This phase-III randomized EBMT-intergroup trial studied the impact of a consolidating autoHSCT vs no consolidation for patients with CLL in Binet stage A progressive, B or C , in CR, nodular PR or VGPR after first or second line therapy. The primary objective was to show that autoHSCT increased the 5-year progression-free survival (PFS) by 30%. Although it had been calculated that 270 patients were to be randomized, the study was terminated by the steering committee in July 2007 due to poor accrual. Here we present a first analysis based on 69% of expected follow-up forms. Results: Between November 2001 and July 2007, 223 patients were enrolled (SFGM-TC/FCLLG n=98, MRC n=62, GCLLSG n=32, SAKK n=10, other EBMT centers n=17). There were 74% males and 26% females. Binet stages were progressive A 13%, B 67%, C 20%; 59% were in CR, and 41% in very good or nodular PR. Of note, SFGM-TC/FCLLG included only patients in CR. 82% of the patients were enrolled in 1st, and 18% in 2nd remission. Patients were randomized between group 1 (autoHSCT n=112) and group 2 (observation n=111) after an induction treatment which was left at the discretion of the investigators. Median PFS was 43 months in the observation group but not reached in the autoHSCT group; 5-year PFS was 48% and 65%, respectively (p=0.005). Accordingly, autoHSCT halved the relapse risk (5-year relapse incidence 25% vs. 51%; HR 0.4 [0.23-0.71], p=0.002). Cox modeling for randomization arm, Binet stage, disease status, line of treatment, contributing group (country), and the interaction between randomization arm and contributing group confirmed that autoHSCT significantly improved PFS (HR 0.41 [0.23-0.75] p=0.004). The beneficial effect of autoHSCT was stable over all contributing groups although patients accrued by SFGM-TC/FCLLG overall had a significantly better PFS than patients from other countries (HR 0.2 [0.08-0.55], p=0.001). At 5 years, the probability of OS was 92% and 91% for autoHSCT and observation, respectively. Significant differences in terms of non-relapse death were not observed. At the last follow up, among 205 evaluable patients, 186 are alive (147CR, 39 relapse), 19 died (14 from relapse and 5 from non-relapse causes) . In conclusion, in patients with CLL in first or second remission, consolidating autoHSCT reduces the risk of progression (PFS) by more than 50%, but has no effect on overall survival. Disclosures: No relevant conflicts of interest to declare.

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Cytogenetic Evolution (CE) In Patients (pts) with Low and Intermediate Risk Myelodysplastic Syndromes (MDS) Is Associated with Poor Prognosis

Blood ◽

10.1182/blood.v116.21.2941.2941 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2941-2941

Author(s):

Liunan Li ◽

Elias Jabbour ◽

Gautam Borthakur ◽

Stefan Faderl ◽

Tapan Kadia ◽

...

Keyword(s):

Disease Progression ◽

Cytogenetic Analysis ◽

Conflicts Of Interest ◽

Myelogenous Leukemia ◽

Intermediate Risk ◽

Hematopoietic Stem ◽

Increased Risk ◽

Significant Difference ◽

The Impact ◽

Cytogenetic Evolution

Abstract Abstract 2941 Introduction: MDS is a spectrum of abnormalities in the proliferation and differentiation of hematopoietic stem cells that result in peripheral cytopenias, bone marrow dysplasia and increased risk of transformation to acute myelogenous leukemia (AML). Cytogenetic abnormalities occur in more than 50% of patients (pts) and have an impact on survival and risk of transformation to AML. CE, or acquisition of additional clonal chromosomal abnormalities, has been reported to occur in 30 to 50% of primary MDS pts. Their impact on prognosis and transformation into AML among pts with low and intermediate risk MDS is not known. In this study, we analyzed the impact of CE on prognosis in lower risk MDS. Methods: we reviewed 722 pts clinic records of low and intermediate risk MDS pts at MD Anderson Cancer Center (MDACC) from 2000–2010 and conducted a retrospective analysis of all MDS pts with at least two consecutive cytogenetic analysis (365 patients, 50.6%) and compared the cytogenetic evolution group (CE group) with the group without cytogenetic changes (no CE group). Cytogenetic analysis was performed in the Cytogenetics Laboratory at MDACC. Results: CE was detected in 200 pts (55%). Characteristics of patients with CE are: median age 65 years (23-91), IPSS int-1 79%, diploid CG 42%, excess blasts 25%. Pts with CE were more frequently female (p=0.005), and had more frequently abnormalities of chromosome 5 and 7 (p<0.001) at baseline. There were no statistically significant difference between these two groups (p>0.05) regarding age, WBC, platelet, hgb, ANC, BM blasts percent, diagnosis (RA or RAEB), and IPSS score. There were more chr.-5/-7, insufficient metaphases, and other abnormalities, but less diploid cases in CE group compared with no CE group (p<0.001). History of malignancy (p=0.001) and prior chemotherapy exposure were also associated with CE (p=0.001), but this was not as strong for radiation exposure (p=0.066). Also, more CE patients required therapy for MDS compared to no CE patients (p=0.039). Progression free survival was significantly extended in no CE patients (p=0.02). Overall survival was a longer in no CE (34.1months), compared with CE group (26.2 months), although this was not statistically significant. Conclusion: CE is more commonly observed among pts with high-risk features, and is usually associated with disease progression and resistance. Also, prior malignancy and chemotherapy exposure were associated with CE in this study. This data indicates that genomic instability has a role in disease progression in MDS. Further analysis of CE in MDS is needed. Disclosures: No relevant conflicts of interest to declare.

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Outcome of Patients with Relapsed/Refractory AIDS-Related Lymphoma In the AIDS Malignancy Consortium (AMC) 1997–2008

Blood ◽

10.1182/blood.v116.21.3569.3569 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3569-3569

Author(s):

Ariela Noy ◽

Ulas Darda Bayraktar ◽

Neel Gupta ◽

Adam M. Petrich ◽

Page Moore ◽

...

Keyword(s):

Conflicts Of Interest ◽

Progression Free Survival ◽

Standard Of Care ◽

Infectious Complications ◽

High Dose ◽

Hiv Diagnosis ◽

Curative Intent ◽

Hematopoietic Stem ◽

The Impact ◽

Aids Related Lymphoma

Abstract Abstract 3569 Introduction: High dose therapy (tx) with autologous hematopoietic stem cell transplantation (AHSCT) in (rel/rfr) lymphoma is the standard of care in the general population with chemosensitive disease. The feasibility of second line therapies (Tx) and AHSCT in (rel/rfr) AIDS related lymphoma (ARL) has been shown in a number of trials. However, the true impact of 2nd line tx and AHSCT is unknown, as nearly all studies focus on those already with disease sensitive to 2nd therapy going onto transplantation. The only recent study capturing patients (n=50) before 2nd line tx showed 49% progression-free survival (Re et al. Blood 2009). Here, we retrospectively analyzed the outcome of patients (pts) presenting at 13 US AIDS Malignancy Consortium sites with (rel/rfr) ARL in the HAART era. Patients and Methods: HIV-positive pts initiating tx for (rel/rfr) ARL between 1997–2008 were included. Overall survival (OS) was calculated from the initiation of 2nd line tx. Results: A total of 126 pts received 2nd line tx. Only those 88 pts who received 2nd line with curative intent to treat (ITT) were included in the analysis. Baseline and selected clinical characteristics are summarized in the table. Median CD4 at HIV diagnosis was 110 (n=37) with a range of 12 to 1000. At ARL dx, median CD4 was 152 (5-803). 47% had an opportunistic infection (OI) prior to ARL. 2nd line tx were: ICE (n=34), EPOCH (n=16), ESHAP (n=11), High-dose MTX variants (n=10), Hodgkin's specific tx (n=5), DHAP (n=4) and others (n=8). Thirty-two (36%) had a response to 2nd line tx (CR, n=21; PR, n=11). Of 50 pts with grade ≥3 toxicities, the most common were thrombocytopenia (46%) and neutropenic fever (44%). Six pts died during 2nd line tx due to infectious complications, with 1 aspergillosis. Best response to 2nd line tx: Thus, CR/PR was 32/88 (36%) in ITT analysis. Only 10/32 CR/PR pts went onto AHSCT due to availability and changing treatment paradigms. Conditioning was BEAM (n=9) and Bu/Cy (n=7). No pt went onto allotransplant. At AHSCT day +90, 10 pts were in CR. For all pts, median follow-up was 122 weeks (range, 8–597), median OS was 38 weeks (95% CI, 27–63). Reflecting the 65% prevalence of pts refractory to 2nd line tx in the non-AHSCT group, OS was longer in pts who underwent AHSCT compared to those who did not (2-year OS: 55.3% vs. 31.0%). Surprisingly, 1-year OS in the CR/PR pts was 87.5±12.5% for AHSCT and 81.8±8.2% for non-AHSCT. One Burkitt pt survived a year without AHSCT. Discussion: Rel/rfr ARL was treated aggressively in this largest ever reported cohort, but CR/PR was only 32/88 (36%) in ITT analysis. Not all CR/PR pts went onto AHSCT due to changing treatment paradigms and regional availability. Aggressive 2nd line tx and ASHCT was feasible despite prior low CD4 and OI, but DFS may be possible without transplant. We cannot draw conclusions about the impact of AHSCT from this retrospective cohort. Similarly, it is not known whether survival in (rel/rfr) ARLs is equivalent to the HIV negative population. The current paradigm is to offer pts with rel/rfr ARLs AHSCT if disease is chemosensitive and no contraindication exist. New strategies are needed for 2nd line therapy, particularly in rel/rfr BL. Disclosures: No relevant conflicts of interest to declare.

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