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We present an individualized systems medicine (ISM) approach to optimize cancer drug therapies one patient at a time. ISM is based on (i) molecular profiling and ex vivo drug sensitivity and resistance testing (DSRT) of patients' cancer cells to 187 oncology drugs, (ii) clinical implementation of therapies predicted to be effective, and (iii) studying consecutive samples from the treated patients to understand the basis of resistance. Here, application of ISM to 28 samples from patients with acute myeloid leukemia (AML) uncovered five major taxonomic drug-response subtypes based on DSRT profiles, some with distinct genomic features (e.g., MLL gene fusions in subgroup IV and FLT3-ITD mutations in subgroup V). Therapy based on DSRT resulted in several clinical responses. After progression under DSRT-guided therapies, AML cells displayed significant clonal evolution and novel genomic changes potentially explaining resistance, whereas ex vivo DSRT data showed resistance to the clinically applied drugs and new vulnerabilities to previously ineffective drugs.Here, we demonstrate an ISM strategy to optimize safe and effective personalized cancer therapies for individual patients as well as to understand and predict disease evolution and the next line of therapy. This approach could facilitate systematic drug repositioning of approved targeted drugs as well as help to prioritize and de-risk emerging drugs for clinical testing.

Contrast enhanced cardiac CT reveals coronary artery disease in 45% of asymptomatic allo-SCT long-term survivors

Technological advances in the laboratory have lead to substantial improvements in clinical decision-making by the use of pre-treatment prognostic risk stratification factors in acute myeloid leukemia (AML). Unfortunately similar progress has not been made in treatment response criteria, with the definition of “complete remission” in AML largely unchanged for over half a century. Several recent clinical trials have demonstrated that higher sensitivity measurements of residual disease burden during or after treatment can be performed, that results are predictive for clinical outcome and can be used to improve outcomes by guiding additional therapeutic intervention to patients in clinical complete remission but at increased relapse risk. We review here these recent trials, the characteristics and challenges of the modalities currently used to detect minimal residual disease (MRD), and outline opportunities to both refine detection and better clinically utilize MRD measurements. MRD measurement is already the standard of care in other myeloid malignancies such as chronic myelogenous leukemia (CML) and acute promyelocytic leukemia (APL). It is our belief that response criteria for non-APL AML should be updated to include assessment for molecular complete remission (mCR) and that recommendations for post-consolidation surveillance should include regular monitoring for molecular relapse as a standard of care.

Long-term survivors of allogeneic stem cell transplantation (SCT) have increased risk of cardiovascular disease. We retrospectively studied cardiovascular risk factors (CVRFs) in 109 SCT survivors (62 males, 47 females; median age 34 years) five years or more after bone marrow (15) or T cell-depleted peripheral blood (94) SCT for CML (56), acute leukemia (29), MDS (13), and others (11). One death and two cardiovascular events were reported. At five and ten years after SCT, respectively, 44% and 52% had abnormal lipid profiles; 23% of 5-year survivors met the Adult Treatment Panel III threshold for dyslipidemia treatment, which is substantially higher than the age-matched general population. There were significant increases in prevalence of hypertension (p0.001), diabetes (p = 0.018), and body mass index (p = 0.044) after SCT compared with baseline. The Framingham general cardiovascular risk score (FGCRS) in males at five years after SCT projected a doubling (median 10.4% vs. 5.4%) in the 10-year risk of cardiovascular events. Females received HRT after SCT, and none had increased FGCRS. Chronic GVHD and C-reactive protein were not associated with CVRF at any time. All CVRFs stabilized between five and ten years after SCT. Thus, SCT survivors have sustained elevations in CVRFs. Males have a significantly increased risk of cardiovascular events in their second and third decade after SCT.

223 Background: To better understand adult acute myeloid leukemia (AML) survivorship and health related quality of life, we piloted a survey exploring patient reported outcomes for patients in first complete remission (CR) to determine if patients feel the survey is relevant to their overall well-being. Methods: A cross-sectional survey measuring: quality of life, physical, role, emotional, cognitive, social functioning (EORTC QLQ-C30 v 3.0); physical, psychological, social, and spiritual well-being (Quality of Life-Cancer Survivor (QOL- CS) scale); fatigue (Functional Assessment of Chronic Illness Therapy – Fatigue Scale (FACIT-Fatigue)); anxiety and depression (Hospital Anxiety and Depression Scale (HADS)); sociodemographic and 5 open-ended questions. Results: 18 participants completed the survey; mean age was 57.2 years. Nine patients were in CR for

The immunotherapy of cancer is arguably the most promising therapy under development, and vaccination against cancer antigens is a promising strategy to stimulate adaptive immune responses against malignant clones. However, the ability of patients treated with chemotherapy to respond appropriately to immune challenge may be suboptimal. This study was designed to determine the functional capacity of the immune system in adult acute myeloid leukemia (AML) patients who have completed chemotherapy and are potential candidates for immunotherapy. We used the response to influenza vaccination as a surrogate for the health of the immune system in AML patients in a complete remission (CR) post-chemotherapy. Ten adult AML patients in CR after completion of intensive chemotherapy were recruited to the clinical protocol J1293. They were on average 37 weeks post-treatment (range 4-148) when receiving the 2012-2013 inactivated seasonal influenza vaccine. Peripheral blood samples were collected at baseline and 30 days post-vaccination. Ten age and sex matched healthy donors (HD) served as baseline controls. Serological response to vaccination was assessed via microneutralization assays; multi-parameter flow cytometry was used to characterize lymphocyte subsets. ELISPOT assays were used to evaluate lymphocyte function, microarrays were used to assess gene expression, and deep sequencing of the B-cell receptor heavy chain (IGH) was performed to determine expansion and clonality of B-cells. Wilcoxon rank-sum tests were used to assess statistical significance. Only 2/10 (20%) patients seroconverted (AML responders, or AML-R) with a four-fold increase or greater in influenza-specific antibody. One responder was 148 weeks post-treatment; the other had acute promyelocytic leukemia (APL) and was 4 weeks post-treatment. Deep immunophenotyping revealed no striking differences in T-cell compartments between AML at baseline and HD, indicating rapid T-cell recovery after chemotherapy. In contrast, we observed a highly atypical B-cell profile. AML non-responders (AML-NR) at day 0 versus HD had significantly reduced frequencies of mature IgA+ (4.5% vs. 11%) and IgG+ (2.4% vs. 5.9%) B-cells (as a percentage of CD19+CD20+). Further dissection exposed markedly higher frequencies of CD10+CD27- transitional B-cells (36% vs. 16%, p Interestingly, functional T-cell assays revealed that of 5 evaluable patients, including the 2 AML-R and 3 AML-NR, 5/5 (100%) patients had an increase in influenza-specific cytokine production (1.24 - 4.40x higher on day 30 over baseline), suggesting a functional T-cell response even with deficient influenza-specific antibody production. Supervised clustering of microarray data identified many upregulated genes in AML-NR related to apoptosis, BCR, IL2, IL-4, IL-8, and IL-12 signaling pathways, indicative of developing B-cells. IGH sequencing demonstrated AML-NR had greater variability in CDR3 length than seen in HD, consistent with an antigen inexperienced B-cell repertoire. These data suggest that while some aspects of cellular immunity recover comparatively quickly, the humoral immune system is incompletely reconstituted in the year following intensive cytotoxic chemotherapy for AML. Abnormal frequencies of transitional and memory B-cells may explain the poor response to vaccination often seen in patients after chemotherapy. Furthermore, the uncoupled recovery of B-cell and T-cell immune capacity observed here might have implications for the success of immunotherapies based on vaccination. Figure 1. Figure 1. Disclosures Noonan: Celgene: Speakers Bureau. Borrello:Celgene: Research Funding.

While commonly used in other hematological malignancies, high dose chemotherapy followed by autologous hematopoietic cell transplantation (auto-HCT) has not been widely adopted in acute myeloid leukemia (AML) due to concerns regarding high post-transplant relapse rates. These relapses may be due, in part, to autograft contamination with AML. High sensitivity methods to detect residual AML have demonstrated the ability to correctly identify patients in morphological complete remission (CR) at risk of relapse. We sought to determine if testing of the graft prior to transplantation could predict post auto-HCT relapse. Prior studies investigating the utility of measurable residual disease (MRD) in auto-HCT have been limited by small patient cohorts and/or by the use of a single biomarker to detect AML, a heterogeneous cancer. We report here the largest retrospective study to date of adult auto-HCT AML patients tested for measurable residual disease (MRD) by both molecular methods (RQ-PCR) and multi-parameter flow cytometry (MPFC). Seventy-two patients transplanted between 2004-2013 at a single academic medical center (UCSF) were eligible for this study based on availability of cryopreserved GCSF mobilized autologous peripheral blood progenitor cell (PBPCs) specimens. All samples were collected on IRB approved research protocols. The median age at time of transplant was 48 (24-69), 54% were female, cytogenetics were known in 94% (of which 24% were favorable, 69% intermediate, 7% poor), 99% were in CR at the time of HCT (64/72 in CR1, 6 in CR2, 1 in CR3). Following auto-HCT 1 year RFS was 50% (1 year relapse rate 43%) and 2 year RFS was 40% (2 year relapse rate 47%). Wilms tumor 1 (WT1) is expressed in up to 90% of AML, but sufficiently over-expressed in peripheral blood to have utility as a sensitive marker of MRD in less than 50% of cases. RQ-PCR detectable WT1 above the previously established European LeukemiaNet threshold was found in 9 patients, of whom 6 relapsed in the first year after auto-HCT (19% sensitivity, 93% specificity, PPV: 67%, NPV: 60%). We have previously reported that multi-gene testing can augment WT1 based MRD detection in AML. We were unable however to use one component of our previously reported AML MRD panel, PR3, due to increased baseline expression in these GCSF stimulated PBPC products. Increased PR3 expression following GCSF administration, correlating with neutrophilia, was also observed in an independent cohort of healthy donors. Testing for PRAME, MSLN, CCNA1, t(8,21), Inv16, t(15:17) and NPM1 mutations A, B and D, as a supplement for WT1, in pre-HCT PBPCs resulted in substantially improved ability to predict post auto-HCT relapse (52% sensitivity, 80% specificity, PPV: 67%, NPV: 69%). Addition of these extra genes allowed for correct identification of 10 additional MRD+ patients who relapsed within 1yr after transplant, and added 5 false positive patients (1 of whom suffered early non-relapse mortality, and another who did relapse but more than 1 year after HCT). Finally, in expert hands, flow cytometry can identify residual AML with high sensitivity. Forty PBPC samples from the above cohort were also assessed for MRD using MPFC. CD34 positive cells comprised 0.05-12.5% of autograft specimen mononuclear cells. Due to immunophenotypic changes likely attributable to GCSF mobilization, and without leukemia associated immunophenotypes from diagnosis available, MPFC was unable to identify MRD in any of 40 patients tested. In summary, no single MRD test on autografts could completely predict post-HCT AML relapse. Auto-HCT presents unique challenges for AML MRD testing due to masking effects of GCSF on MPFC and RQ-PCR gene expression signatures. Additionally, detection of any AML autograft contamination must be extremely sensitive to be useful for predicting relapse given the absence of any potentially protective graft versus leukemia effect following an auto-HCT. Here we show combinations of molecular MRD assays can overcome some, but not all, of these limitations (Figure). Figure 1. Figure 1. Disclosures Radich: Incyte: Consultancy; Ariad: Consultancy; Novartis: Consultancy; Novartis: Other: Lab Contract. Andreadis:Cellerant: Consultancy; Novartis: Consultancy; McGraw Hill: Other: Publishers; Pharmacyclics: Honoraria. Damon:Sunesis: Research Funding; McGraw Hill: Other: Chapter Royalties; Atara: Consultancy; Sigms Tau: Research Funding. Logan:Jazz Pharmaceuticals: Consultancy; Amgen: Consultancy; Pharmacyclics: Consultancy. Martin:Sanofi: Consultancy.

There is increasing recognition of the role of inherited germline predisposition for myeloid disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The additional somatic genetic events required for development of a malignant phenotype are however poorly understood. A 25 year old woman was referred to the NHLBI hematology branch in March 2014 for a seven year history of pancytopenia. Her medical history included recurrent pneumonias, oral ulcers, severe varicella infection and arthralgias. Prior bone marrow examinations at ages 21 and 23 at outside institutions reported normocellular marrow, tri-lineage hematopoiesis and mild dyspoiesis. Cytogenetics were remarkable for trisomy 8 in 80% (aged 21) or 90% (aged 23) of metaphases. Previously unrecognized lymphedema was noted on examination. Peripheral blood counts showed WBC 2.28 K/ul [normal range: 3.98-10.04], HGB 9.9 g/dL [11.2-15.7], PLT: 67 K/ul [173-369], ALC: 0.36 K/ul [1.18-3.74] and AMC: 0.06 [0.24-0.86]. Peripheral blood flow cytometry demonstrated decreased CD3+ CD4+ (T) cells, CD19+ (B) cells and NK cells. HLA-DR15 negative. Bone marrow examination showed trilineage hematopoiesis, 50-60% cellularity, mild erythroid predominance and mildly increased, mildly atypical megakaryocytes. Blasts less than 5%. Bone marrow flow cytometry revealed severely decreased B-cells and monocytes, absent B-cell precursors, absent dendritic cells, inverted CD4:CD8 ratio, and atypical myeloid maturation pattern. Cytogenetics demonstrated stable trisomy 8 in 90% of metaphases. On the basis of this assessment the diagnosis of MDS was confirmed. Sanger sequencing revealed a GATA2 L375S mutation in the second zinc finger of known pathogenic significance. Four months later she developed increased fatigue and easy bruising with worsening thrombocytopenia (PLT: 10K/ul). Bone marrow was dramatically changed; now markedly hypercellular (90-100%) with diffuse sheets of immature cells consistent with blasts having fine chromatin, distinct or prominent nucleoli, and visible cytoplasm. Blasts were positive for CD33, CD56, CD64, CD123, and CD163; and were negative for CD34, CD14, and myeloperoxidase. Cytogenetics showed a new trisomy 20 in 65% of metaphases, in addition to previously seen trisomy 8 in 100%. A diagnosis of acute monoblastic leukemia (M5a subtype) was made. At both clinic visits bone marrow aspirate was collected on an IRB approved research sample acquisition protocol. Whole exome sequencing of 1ug DNA was performed using Agilent SureSelect v5 Exome enrichment Kits on an Illumina HiSeq 2000 with 100-bp paired-end reads (Macrogen, Rockville, MD). Data was mapped to hg19 (BWA) and processed using an in-house pipeline (Samtools/Picard/GATK/VarScan/Annovar). Mean read depth of target regions was 157 and 149. There was high correlation between both samples with the exception of a NRAS:NM_002524:exon3:c.C181A:p.Q61K mutation (57 of 180 reads) seen only in the later sample. Confirmatory ultra-deep sequencing for NRAS was performed using Illumina TruSight Myeloid Sequencing Panel on an Illumina MiSeq. No evidence of the NRAS Q61K mutation was found in the earlier March MDS bone marrow sample even when sequenced to a depth greater than 1750 reads (see figure). The mutation was confirmed in the August AML sample at a variant allele frequency of 35%. If heterozygous this would reflect a clone size of 70%, consistent with data from both cytogenetics (new trisomy 20 in 65% of metaphases) and the 76% blasts documented by bone marrow aspirate smear differential. We report here the rapid progression to AML in a patient with germline GATA2 MDS associated with development of a new trisomy 20 karyotype and a NRAS Q61K mutation. The NRAS mutation was not detectable after the patient achieved a complete remission following induction chemotherapy further supporting this association. This NRAS mutation has been implicated in the pathogenesis of multiple cancers by constitutive activation of proliferative signaling. GATA2 associated MDS is a high-risk pre-leukemic condition with the potential for rapid evolution to AML. This is the first report of acquired somatic mutations in the RAS/RTK signaling pathway in the context of germline GATA2 insufficiency associated with acute leukemic transformation. Figure 1. Figure 1. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding.

INTRODUCTION: Relapse is the most important cause of post-transplant mortality. The interaction between killer immunoglobulin-like receptors (KIRs) of donor natural killer (NK) cells and human leukocyte antigen (HLA)-class I molecules on recipient target cells may influence the outcome of allogeneic hematopoietic stem cell transplantation (HCT) by modulating NK cell alloreactivity. In addition to modulation by KIRs, NK cells also respond to CMV reactivation post-HCT to promote maturation and functional competence that could enhance their antileukemic effect. Since NK cells are postulated to play a prominent role in the setting of ex vivo T cell depleted HCT, we studied the relative contributions of KIR-mediated alloresponse and early CMV reactivation, in addition to measurable residual disease (MRD) status, on clinical outcomes. METHODS: A cohort of 109 consecutive patients who received myeloablative, HLA-identical sibling allogeneic peripheral blood HCT for hematological malignancies was studied at a single institution. All patients received a myeloablative preparative regimen (fludarabine 125 mg/m2, cyclophosphamide 120mg/kg, total body irradiation 400-1200 cGy based on age) followed by the infusion of a CD34+ selected graft from an HLA-identical sibling. Three models of donor KIRs were evaluated: a) KIR2DS1 positive (Venstrom, et al. NEJM 2012) seen in 41% of donors, b) "KIR3" - all 3 KIRs 2DL5A, 2DS1, and 3DS1 (Stringaris, et al. BBMT 2010) seen in 34% or, c) any KIR B genotype defining loci (KIR2DL5, 2DS1, 2DS2, 2DS3, 2DS5, or 3DS1) seen in 68%. CMV reactivation (65%) was described as a time-dependent covariate as well as dichotomized into early (< Day 60) vs other. Baseline multigene array MRD status was determined by RT-PCR (Goswami, et al. BMT 2015) and found to be positive in 6/21 (29%) of AML patients. Cox proportional hazards models were used. The cumulative incidence of relapse (CIR) was estimated and compared by the Gray's method to account for competing risks. RESULTS: Median recipient age was 43 years and median HCT-CI score was 3 (range 2 - 4). 40% of the cohort had AML, 26% ALL, 19% MDS, 10% NHL/CLL and 5% CML. At a median follow up of ~ 5 years, cumulative incidence of relapse was 35.4%, overall survival (OS) was 48.3% (95% CI 39.2-59.5), and NRM was 26.5%. CMV reactivation was not significantly associated with CIR as a time-dependent covariate (HR 1.4, P =0.35) or as a dichotomized covariate (HR 1.7, P =0. 16). Donor KIR2DS1 (HR 1.8, P = 0.07), KIR3 positivity (HR 1.9, P =0.06), and any haplotype B (HR 1.5, P =0.25) were not significantly associated with CIR. In the multivariate models adjusted for age at HCT, disease risk and CMV reactivation. However, MRD status in AML subjects was highly predictive of future relapse (MRD+ CIR was 83% vs MRD- CIR 21%, HR 7.5, p = 0.0015)(Figure 1). CONCLUSIONS: Pretransplant MRD (confined to AML subjects) strongly predicted future AML relapse in our models, and there was no significant association found with early CMV reactivation or with donor KIRs. Figure 1. Cumulative Incidence of Relapse by MRD Status. Figure 1. Cumulative Incidence of Relapse by MRD Status. Disclosures No relevant conflicts of interest to declare.

Hematologic malignancies were the first diseases in clinical oncology for which the potential of harnessing the immune system as targeted therapy was unequivocally demonstrated. Unfortunately, the use of this highly efficacious modality has been limited to only a subset of patients and diseases because of immune-mediated toxicities resulting from incomplete specificity, and disease-specific determinants of sensitivity versus resistance to immune effector mechanisms. Recent studies, however, have begun to elucidate the molecular basis of the observed clinical effects allowing the rational development of next generation of immunotherapeutic combinations. We discuss here cancer antigen targets in hematologic malignancies and the specific approaches to induce immunity being pursued, the importance of modulating the host immunoregulatory environment, and the special features of immunological monitoring in clinical investigation. The hematologic malignancies represent an ideal setting for the development of immunotherapy due to logistical, clinical monitoring, and disease biology factors and may represent an exemplar for immune-based treatment in other cancer types.

Introduction Incidence of oesophageal adenocarcinoma (OAC) is increasing rapidly. OAC arises in columnar-lined oesophagus (CLO), a metaplastic change affecting some patients with gastro-oesophageal reflux disease (GORD). As yet there is no reliable method of identifying those at highest risk. Our earlier observation of an association between OAC and blood group O Rhesus negative, if confirmed, may help identify those at greatest risk.Aim and methods To assess the distribution of blood group and Rhesus D (RhD) factor in patients with GORD compared with the blood donating general population. GORD was categorized as nonerosive reflux (NER), erosive oesophagitis, CLO and OAC. The Rotherham Hospital database holds details of all GORD, CLO and OAC patients seen in the Gastroenterology Unit. Blood group information for patients with GORD was obtained from patients' records and the hospital's blood transfusion service. The blood group distribution in the general population was obtained from the National Blood Transfusion Service. The number of expected to observed patients in each blood group for each subtype was compared.Results Two thousand six hundred and ten NER, 2813 erosive oesophagitis, 568 CLO and 73 OAC patients had a recorded blood group. For RhD positive patients observed proportions in each blood group were similar to expected. The most striking difference was the marked excess of OAC in blood group O, Rhesus negative (P = 0.002).Conclusion CLO patients with blood group O, RhD negative carry a disproportionately higher risk of developing OAC. The mechanism is unknown but the finding has practical application in guiding risk stratification and intensity of surveillance. Eur J Gastroenterol Hepatol 23: 801-806 (C) 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Acute myelogenous leukemia (AML) is a disease more common in older patients than in the young. It is increasingly recognized that conventional cytotoxic chemotherapies used in children and young adults may not be appropriate in older adults because of diverse host- and disease-biology factors. This review highlights some of the most promising new treatment options that are being evaluated for older patients with AML. These options include CPX-351 (Celator Pharmaceuticals Inc), a unique liposomal formulation of a fixed ratio of cytarabine and daunorubicin; timed sequential therapy with the CDK inhibitor alvocidib (flavopiridol; sanofi-aventis/NCI); the second-generation purine nucleoside analog clofarabine; the farnesyltransferase inhibitor tipifarnib (JohnsonJohnson Pharmaceutical Research and Development LLC); and the DNA methyltransferase inhibitors decitabine and azacitidine.

Ex vivo T cell depletion (TCD) of the graft by CD34+ selection improves the tolerability of allogeneic hematopoietic stem cell transplantation (HCT). Clinical comorbidity measures enhance the estimates of HCT tolerance and outcomes. However, clinical prediction tools such as the hematopoietic cell transplantation-specific comorbidity index (HCT-CI) have not been validated in TCD transplants. To improve outcome prediction we therefore evaluated published comorbidity measures and other standard clinical biomarkers of outcome in a series of patients with hematological malignancies receiving myeloablative TCD transplants. Pre-transplant factors compared were: HCT-CI, European Group for Blood and Marrow Transplantation (EBMT) risk score, Eastern Cooperative Oncology Group (ECOG) Performance Status, serum C-reactive protein (CRP), albumin (ALB), pre-albumin (PAB), ferritin, absolute lymphocyte counts (ALC), and absolute lymphocyte / monocyte ratio (LMR). All patients received a myeloablative preparative regimen (Flu 125 mg/m2, Cytoxan 120mg/kg, TBI 400 cGy if older than 55 or 1200 cGy if younger than 55) followed by the infusion of a CD34+ selected graft from a HLA-identical sibling containing 1 to 5x10e4 CD3+/kg. Of the 79 recipients, 34 (43%) had standard risk disease, and 45 (57%) recipients had high-risk disease. At a median follow up of ~ 5 years, overall survival (OS) was 42.9% and nonrelapse mortality (NRM) was 32.9%. In the initial analyses we eliminated pre-transplant ECOG, ferritin, ALB, PAB, ALC and EBMT. The independent pre-transplant comorbidity variables identified for further testing were HCT-CI, CRP and LMR. We demonstrated that LMR and HCT-CI score are important independent clinical predictors of OS and NRM in TCD HCT. In univariate analysis of OS, significant factors were HCT-CI scores ≥ 5 (Hazard ratio (HR) 2.125, p= 0.018), CRP (HR=1.016, p=0.058, a trend) and LMR ≤ 1.3 (HR=2.054, p= 0.036). In multivariate models of OS, CRP (HR 1.02, p= 0.029), HCT-CI≥5 (HR 1.966, p= 0.04) and LMR ≤1.3 (HR 2.206, p= 0.029) retained significance. Further refinement of the multivariable model was made by classifying the LMR into a low-risk group (LMR 3-25) versus high-risk groups (LMR< 3 or > 25), then the significant factors were HCT-CI ≥5 (HR 3.008, p= 0.002), LMR < 3 (HR=3.292, p= 0.0014) and LMR > 25 (HR=5.648, p= 0.0008) compared to the LMR 3-25 group. In multivariable analysis of NRM, significant factors were HCT-CI ≥5 (HR 3.283, p= 0.015), LMR < 3 (HR=5.891, p= 0.0008), and LMR > 25 (HR=9.008, p= 0.0011) compared to the LMR 3-25 group. To evaluate the relevance of these factors in predicting the 5-year mortality and NRM beyond traditional risk indicators, we used the continuous net reclassification improvement (NRI) and the C-statistic (area under the receiver operating characteristic (ROC) curve). LMR was found to be the most important risk predictor for both OS and NRM (NRI was 84% and the C-statistic was improved from 0.59 to 0.68 with the addition of LMR to the multivariate model for OS; NRI was 108% and the C-statistic was improved from 0.57 to 0.72 with the addition of LMR to the multivariate model for NRM). Based on our results, we propose a simple novel predictive score for CD34+ selected myeloablative transplantation calculated by summation of scores derived from the HCT-CI [≤4 =Score 0, ≥5=Score 1] and LMR [3-25= Score 0, 25=Score 2]. The scores [range, 0 to 3] differentiate three risk groups for hematopoietic cell transplantation OS (p=0.001) and NRM (p=0.004). In conclusion, this is the first study to explore pre-transplant comorbidity measures after TCD HCT, identifying HCT-CI and LMR as critical predictors of OS and NRM. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background. Emerging evidence indicates that microRNAs (miRNAs) may regulate immunity. Their role in acquired aplastic anemia (AA), a T cell-mediated autoimmune disease, has not been investigated. We characterized miRNA signatures in patient T cells to identify novel genes involved in the regulation of immune responses of CD4+ and CD8+ T cells in AA. Cells from age-matched healthy donors and patients with myelodysplastic syndrome (MDS) and transfusion-dependent sickle cell disease (SCD) were used for comparison. Method. MiRNAs and mRNAs from flow cytometrically sorted T cells from patients and controls were extracted and subjected to profiling using miRNA PCR array platform (miScript miRNA PCR array, MIHS-111Z, Qiagen Inc). Real-time PCR was employed for validation of specific miRNA expression changes. To define identified miRNA functions, their target genes were predicted using bioinformatic software (TargetScan, miRBase, DIANA-miRPath). MiRNA target gene profilings were performed using custom PCR array platform (Custom PCR array, Qiagen Inc) in CD4+ and CD8+ T cells in AA. MiRNA knockdown (miR-126-3p and/or miR-145-5p) on healthy CD4+ and CD8+ T cells was performed using the Human T Cell Nucleofector kit (Lonza AG). Cell proliferation was estimated by CFSE assays. Effects of miRNA knockdown on the target genes were evaluated by qPCR and immunoblot. MiRNA expression profilings were examined in CD4+ and CD8+ T cells in a mouse model of lymph node cell infusion-induced bone marrow failure (BMF). Results and Discussion. As compared to healthy controls, miRNA expression profiling revealed significant down-regulation of miRNAs in a disease-specific pattern. MiR-199a-5p expression was reduced in CD4+ and CD8+ T cells from patients with all diseases that were tested (AA, MDS, SCD). Reduced expression of miR-223-3p (more than 3 fold change (FC), P Conclusion. This work describes previously unknown potentially regulatory role of miRNAs 145-5p and 126-3p in T cell activation in AA. Further investigation is warranted to determine the role of these miRNAs in the more general pathogenesis of autoimmune diseases. Understanding these novel pathways might offer new therapeutic strategies and a novel mechanistic insight into the epigenetic control of human T cell effector responses. Figure 1 Figure 1. Disclosures Townsley: GSK: Research Funding.

Oral Mucositis (OM) is a frequent and debilitating complication of standard myeloablative T-replete allogeneic stem cell transplantation (allo-SCT), often requiring narcotic analgesia, total parenteral nutrition (TPN) and prolonged hospitalization. The incidence of World Health Organization (WHO) grade 3-4 OM after total body irradiation (TBI) based myeloablative conditioning can exceed 50% when methotrexate is used for graft versus host disease (GVHD) prophylaxis. Ex vivo T cell depleted (TCD) allo-SCT is known to be associated with improved GVHD-free survival. However its impact on early transplant outcomes, particularly the incidence of OM has not been described. We conducted a single institution retrospective analysis of the severity and incidence of OM and its related outcomes in 105 consecutive recipients of ex vivo T cell depleted (TCD) allo-SCT from their 6/6 matched sibling donors between 2006 to 2014. All subjects received a fully myeloablative conditioning regimen consisting of fludarabine (25mg/m2/day) from day -8 thru day -4, age-adapted fractionated TBI from day -7 thru day -4 followed by cyclophosphamide (60 mg/kg/day) on day -3 and day -2. TBI was delivered in 8 fractions at a total dose of 1200cGy with lung shielding to 600cGy for subjects Disclosures No relevant conflicts of interest to declare.

Background: Acute myeloid leukemia (AML) is a diagnosis encompassing a diverse group of myeloid malignancies. Heterogeneous genetic etiology, together with the potential for oligoclonality within the individual patient, has made the identification of a single high sensitivity marker of disease burden challenging. PCR based assessment of WT1 expression has been extensively tested as a marker of submicroscopic AML disease burden but is not universally overexpressed in all cases. Our hypothesis was that the addition of other individually suboptimal gene expression assays to the backbone of WT1 PCR based testing in a multi-gene array format would improve prediction of relapse and survival when tested prior to allogeneic stem cell transplantation (allo-SCT). Patients and Methods: We identified pre-transplantation peripheral blood samples from 74 AML patients who had received allo-SCT as part of NHLBI clinical protocols performed at the NIH clinical center in Bethesda, Maryland between 1994 and 2012. All had morphological evaluation of disease status within the two months prior to transplantation and at least twelve months of documented post-SCT clinical outcomes data or until date of death if this occurred before twelve months. Transplantswere predominately myeloablative, T cell depleted, peripheral blood stem cell sibling matched allogeneic transplants with cyclosporine-based graft versus host disease prophylaxis. Peripheral blood samples from fifty healthy adults donors were used as baseline controls. All clinical protocols and permission to use blood for research were conducted in accordance with Declaration of Helsinki principles and were approved by the Institutional Review Board with written informed consent obtained from all subjects. One microgram of total RNA isolated from cryostored peripheral blood samples was reverse-transcribed into cDNA and used for quantitative real time PCR (qRT-PCR) reactions using custom multi-gene PCR array plates (MG-MRD) and SYBR green, with normalization to c-abl. Arrays included primers for detection of WT1, CCNA1, PRTN3, PRAME, MSLN and ABL transcripts. Thresholds for positivity were established based on expression levels seen in healthy normal donors. Statistical analysis was performed using GraphPad Prism (La Jolla, CA, USA). Comparison between survival or relapse curves was performed using the Log-rank (Mantel-Cox) test. Results: In this cohort of 74 patients the overall survival observed at three years after transplant (3yr OS) was calculated to be 36%. Traditional response criteria from examination of bone marrow risk stratified these 74 patients into two groups, the morphological CR group (n=48) with a 3yr OS of 48% and a group of 26 patents with active (refractory/refractory) disease at the time of allo-SCT with a 3yr OS of only 15%. Peripheral blood testing for WT1 overexpression identified 25 patients as positive (3 of whom had no clinically evident disease by morphology on bone marrow examination, ie; minimal residual disease, MRD). Testing using the MG-MRD assay identified an additional 9 patients from the WT1 negative group as also having residual disease (2 had morphologically evident disease, 7 with MRD). The mortality in this group of patients following transplantation was 100%. The 34 (of the total 74) patients with positive MG-MRD prior to transplantation had a three year overall survival of just 9%. This MG-MRD positive group also experienced 22 out of a total of 24 relapses observed in the first year following transplantation. Pre-SCT MG-MRD testing correctly predicted all cases of early relapse (ie: within 100 days after allo-SCT) compared to just 57% (8/14) using WT1 alone. For the 37 patients who entered transplant in a pathological CR, and who did not suffer non-relapse mortality in the first year, the 1yr relapse rate was 24%. Pre-SCT WT1 testing had 100% positive predictive value (PPV) but only 33% sensitivity in identifying relapses in this subgroup, compared to 100% PPV and 89% sensitivity for the MG-MRD array. Conclusion: PCR based pre-SCT MRD testing using a multi-gene panel outperformed WT1 alone, correctly identifying 36% more patients (n=9, all of whom died) as belonging to a group at high-risk for post-SCT relapse and death. Collaborations are now being sought to validate these findings in additional cohorts. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Adult acute lymphoblastic leukemia (ALL) remains a therapeutic challenge with less than 40% long term survival. There is growing evidence that malignant diseases exert an “immune editing” effect which blocks antitumor immunity and permits tumor growth through immune evasion. Such tumor escape represents an obstacle for anticancer immunotherapy. In ALL such immune escape mechanisms are not well characterized. We therefore profiled cellular immunity in ALL, by characterizing the subsets of T cells, regulatory T cells (Treg), natural killers (NK) cells and γd T cells, using various functional markers including T cell exhaustion and NK cell activating or inhibitory molecules. Forty ALL patients were included in the study. The median age was 39 y (range, 18-75). Thirty-six presented with B-lineage ALL and 4 with T-lineage ALL. Mononuclear cells were isolated from blood (n=19) or bone marrow (n=21) at the onset of leukemia or at relapse. The median infiltration of blasts was 85% (range 24-96%). Healthy donor peripheral blood (n=12) and bone marrow (n=9), from age and gender matched population, were simultaneously analyzed as controls. Extra-and intra cellular staining were performed using using antibodies directed against CD3, CD4, CD8, CD45, CD45, CD45RA, CD45RO, CCR7, CD95, CD27, CD19, CD14, CD127, CD25, Foxp3, Helios, αβTCR, HLA-DR, CD117, CD20, CD10, CD22, CD34, LAG3, PD1, PDL1, CD56, NKG2A, NKG2C, NKG2D, KIR2DL1, KIR2DL3, CD57, CD33, CD11b, CD15, CD38 and CD24. Data were acquired on a BD LSRFORTESSA flow cytometer. The expression of programmed cell death 1 (PD-1, CD279) receptor on CD8+T cells was significantly increased in blood and bone marrow of ALL patients compared to healthy donors (p Adult patients with ALL show evidence of immune-editing of T cells and NK cells. This global immunosuppressive mechanism may contribute to the eventual escape of ALL from immune control. PD-1, overexpression, described in acute myeloid leukemia and chronic myeloid leukemia has been implicated in T-cell exhaustion and subsequent tumor immune evasion. Our data suggests similar immune escape mechanisms pertain in ALL. Effective antileukemia immunotherapy will require targeting one or more of these immunosuppressive pathways to achieve optimum results. Disclosures Fathi: Seattle Genetics, Inc.: Consultancy, Research Funding; Takeda pharmaceuticals International Co.: Research Funding; Exelixis: Research Funding; Ariad: Consultancy.

Outcomes for acute/leukemic adult T-cell leukemia/ lymphoma (ATL) remain extremely suboptimal [1,2]. Median survival of only 9 months and 5-year overall survival rates of less than 30% have recentl...

The immune-editing effect of myeloid leukemia has recently been reported in several studies. We previously demonstrated that the K562 leukemia-derived cell line suppresses T cell proliferation, which suggests that myeloid leukemia may function in a similar way to myeloid derived suppressor cells (MDSC). While the mechanism of suppression in leukemia is not fully understood, recent murine and human studies suggest that the STAT3 and arginase pathways play a key role in the immunosuppressive function of MDSC. We hypothesized that myeloid leukemia utilizes the MDSC STAT3 and arginase pathway to evade immune control, and block anti-leukemic immune responses. To evaluate the suppressive capacity of myeloid leukemia on T cell proliferation, we isolated CD34+ blasts and myeloid derived suppressor cells (MDSC: CD11b+CD14+) from blood of primary leukemia samples by FACS sorting (n=5). These cells were co-cultured with CFSE-labeled CD4+ T cells (n=9), previously isolated from healthy donor PBMCs using an automated cell separator (RoboSep). After stimulating with CD3/CD28 Dynabeads (Invitrogen, New York, USA) for 72 hours, proliferation was measured by CFSE dilution of the viable cell population. In three myeloid leukemias studied, CD4+ T cell proliferation was significantly suppressed in the presence of primary CD34 blasts and MDSC cells (p0.05), consistent with the results obtained with the primary leukemia samples. To evaluate the impact of STAT3 and arginase on the immunosuppressive function of myeloid leukemia, the five cell lines were primed overnight with either arginase inhibitor (N(ω)-Hydroxy-nor-L-arginine; EMD Biosciences, Inc., California, USA) or two STAT3 inhibitors (STAT3 Inhibitor VI or Cucurbitacin I; EMD Millipore, Massachusetts, USA). Then, CD4+ T cells from healthy donors (n=3) were cultured with either (1) leukemia without any inhibitor (2) leukemia in the presence of inhibitor (3) leukemia primed with inhibitor. Priming leukemia with arginase inhibitor and STAT3 inhibitors almost completely abrogated their suppressive effect of T cell proliferation (p We conclude that myeloid leukemia, like MDSC, directly immunosuppresses T cells, through STAT-3 and arginase. This finding may underlie the immune-editing of T cells by myeloid leukemia. Our results suggest that STAT3 inhibitors could be used to augment leukemia-targeted immunotherapy. Further investigation of T cell biology within the leukemia microenvironment is needed to further define immune editing mechanisms in myeloid leukemia. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures: No relevant conflicts of interest to declare.

Introduction It is now clear that traditional response criteria in acute myeloid leukemia based on morphological assessment alone is suboptimal for the prediction of long-term outcome following treatment. While pre-treatment risk stratification based on cytogenetic and molecular correlates of disease biology is highly informative, direct assessment of disease burden after treatment while in traditional complete remission can provide additional prognostic information. Determination of minimal residual disease (MRD) can be performed by a variety of methods; in AML the absence of a unique marker as found in CML and APL has arguably limited the clinical adoption of this higher sensitivity standard. Wilms tumor-1 (WT1) is a commonly over-expressed gene in AML proposed as a standardized MRD assay; unfortunately it is not expressed in 10-20% cases of AML, and is overexpressed to a level necessary for high sensitivity measurements of disease burden in only around 50% of cases. We performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) to determine the magnitude of overexpression of 65 genes using 30 peripheral blood samples from untreated AML patients and 50 healthy normal donor (ND) controls. From this analysis we selected 11 genes with sufficient overexpression for potential use in AML MRD assessment. We tested the predictive and prognostic capacity of this novel multi-gene array to predict hematological relapse using peripheral blood samples from 18 AML patients either prior to, and/or in the surveillance period following, allogeneic stem cell transplantation (SCT). Methods De-identified, clinically annotated peripheral blood samples from AML patients who had received allogeneic, non-myeloablative, T-cell depleted SCT from 2004 -2012 collected under IRB-approved protocols at the National Institutes of Health Clinical Center were studied. Expression of 11 genes was assessed using our custom-designed qRT-PCR array, normalized to ABL1 expression and analyzed using the comparative ddCT method After excluding one outlier for each gene, the threshold for positivity was set as the upper limit observed in ND controls. In serial samples, molecular relapse was defined as new gene overexpression seen on two consecutive time-points with the time of relapse defined as first detection of elevation plus 7 days to allow for retesting. Results Eighteen patients were studied retrospectively. Average age was 41 years (range 17-72) with 10 female and 8 male. Sixteen patients had available pre-transplant peripheral blood with a minimum clinical follow-up of 10 months and 14 patients had at least 6 months of follow-up samples (including a sample in the three months prior to clinical relapse if applicable). In samples from 16 patients taken prior to SCT, WT1 overexpression was detectable in 9 of the 12 patients who relapsed within ten months after SCT and in none of 4 non-relapsing patients (sensitivity: 75%, specificity: 100%, PPV: 100%, NPV: 57%). In comparison, the use of three genes (WT1, CCNA1 and HBG2) from our multi-gene array correctly predicted 11 of these 12 relapsing patients and was also negative in all non-relapsing patients (sensitivity: 92%, specificity: 100%, PPV: 100%, NPV: 80%). For those nine patients judged to be in clinical remission at the time of SCT, the use of WT1 overexpression alone as a marker of MRD predicted only three of the six relapses in the following ten months (sensitivity: 50%, specificity: 100%, PPV: 100%, NPV: 50%) compared to the identification of five of the six relapses (sensitivity: 83%, specificity: 100%, PPV: 100%, NPV: 75%) using our three gene pre-SCT signature. In serial surveillance samples from 14 patients following SCT, 7 of the 11 relapsing patients were detected by WT1 overexpression alone compared to 9 detected using our multi-gene signature (sensitivity 64% vs. 82%), with no false positives detected using either test (specificity 100% and PPV: 100% for both methods). The mean time between detection of molecular relapse and clinical diagnosis using WT1 testing was 24 days (range 0-79 days) compared to 46 days (range 0-179 days) using our multi-gene array. Conclusion Assessment of minimal residual disease for AML using our multi-gene array is highly sensitive and may improve the ability to predict or detect relapse in both the pre and post SCT settings compared to assays of single gene overexpression. This array will now be tested prospectively. Disclosures: No relevant conflicts of interest to declare.

Between presentation and remission of AML, loss of leukemia burden and the recovery of normal hematopoiesis are likely to be associated with major changes in cytokine profiles which could inform pathophysiology of hematopoiesis and immune recovery and may be predictive for outcome. However cytokine fluctuations in AML before and after induction chemotherapy are not well characterized. To profile the cytokine signatures of patients with AML, we analyzed 57 cytokines, chemokines, and growth factors in blood of 11 patients with AML (mean age 58 years; 31-69) undergoing conventional remission induction chemotherapy enrolled into an investigational study (VICCHEM 1073). Plasma was obtained from heparinized peripheral blood collected at onset of leukemia, 8-14 days, and 22-35 days after the initiation of induction chemotherapy and also from 12 healthy donors. Cytokine levels were measured in duplicate using magnetic beads based Luminex assay (Affymetrix, CA, USA). Compared with normal controls, 5 cytokine patterns were observed. i) levels significantly lower at onset of leukemia, and lowest 8-14 days after induction correlating with lymphocyte count: GM-CSF, M-CSF, PDGF-AA, EGF, FGF basic, IL1b, IL-2,IL4, IL10, IL12p40, IL12p70, IL13, IL15, IL17a, IL22, IL23p19, TNF beta, TNF alpha, IFN alpha, IFN gamma, TGF alpha, MCP3, LIF, Granzyme B, sFAS ligand, TRAIL, (p Disclosures: No relevant conflicts of interest to declare.

In a subgroup of patients with myelodysplastic syndrome (MDS) an immune mechanism is believed to cause bone marrow failure. In these individuals immunosuppressive treatment (IST) can restore marrow function. We previously reported a 68% response rate in a phase I/II study of alemtuzumab in 31 patients with low to int-2 risk MDS (Sloand etal JCO Dec 10, 2010:5166-5173). Patients were enrolled according to their predicted probability of response based on an algorithm that incorporated HLA-DR15 positivity, age and transfusion burden (Blood 2003 Oct 15; 102(8):3025-7). We now report updated efficacy data on our cohort of 40 patients and long term follow up data on responders. The first 37 patients received 10mg of alemtuzumab for 10 days intravenously. Alemtuzumab was administered subcutaneously in the subsequent 3 patients. Primary endpoints were changes in peripheral blood counts. Secondary endpoints included improvement in the transfusion requirements (in transfusion- dependent patients), duration of response, and late effects of treatment, relapse and survival. Thirty nine patients were evaluable. One patient discontinued treatment after one dose because of severe hypotension and was excluded from analysis. Median follow-up time was 25 months (3-64 months). Median age was 56 years (23-71 years). The overall response rate (ORR) was 64% (25/39) with 21% of patients having a complete response (8/39). Twenty of 29 (69%) int-1 patients; 4 of 7 (57 %) int-2 patients and 1 of 3 (33%) low risk patients responded following alemtuzumab treatment. Median time to response was 3 months with median duration of response 9 months (range 1-69 months). Eight of 11 patients who relapsed regained their response with addition of cyclosporine and one patient was lost to follow up. The median duration of response in patients rescued with cyclosporine is 37 months (1-42 months). The median duration of response to immunosuppressive treatment (alemtuzumab +/- cyclosporine) is 22.2 months (range 3-69 months). All responding patients who were previously transfusion dependent became transfusion independent after alemtuzumab treatment. One of three patients who received subcutaneous alemtuzumab administration responded. The median survival for all patients was 34 months (range 1-72 months) (figure 1a) with median survival of 36 months (6-72 months) in responding group compared to 16 months (1-56 months) in non-responding group (p Disclosures: Off Label Use: Alemtuzumab use as immunosuppresive agent in myelodysplastic syndrome.

The ability to target myeloid leukemia with immunotherapy would represent a significant therapeutic advance. We report here immunological analysis of clinical trials of primary and secondary vaccination with K562/GM-CSF immunotherapy in adult chronic phase chronic myeloid leukemia patients (CML-CP) with suboptimal responses to imatinib mesylate. Using serological analysis of recombinant cDNA expression libraries of K562 with autologous vaccinated patient serum, we have identified 12 novel chronic myeloid leukemia-associated antigens (LAAs). We show that clinical responses following K562/GM-CSF vaccination are associated with induction of high-titer antibody responses to multiple LAAs. We observe markedly discordant patterns of baseline and induced antibody responses in these identically vaccinated patients. No single antigen was recognized in all responses to vaccination. We demonstrate that an additional ‘booster’ vaccination series can be given safely to those with inadequate responses to initial vaccination, and is associated with more frequent induction of IgG responses to antigens overexpressed in K562 vaccine compared with primary CML-CP. Finally, those with induced immune responses to the same LAAs often shared HLA subtypes and patients with clinical responses following vaccination recognized a partially shared but non-identical spectrum of antigens; both findings have potentially significant implications for cancer vaccine immunotherapy.

3104 Background: The ability to target myeloid malignancies using immunotherapy, without allogeneic transplantation, depends on the capability to target leukemic clones while sparing normal tissues. A variety of putative leukemia associated antigens (LAA) have been identified but an evidence-based list of targets for acute myeloid leukemia (AML) has not yet been established. Methods: De-identified, clinically annotated, samples of peripheral blood and/or bone marrow aspirate from untreated AML patients were collected under IRB-approved protocols from three NCCN cancer centers. Samples were analyzed for commonly observed somatic mutations in ASXL1, DNMT3A, FLT3, IDH1/2, KIT, NPM1, NRAS, RUNX1, TET2, and WT1. Gene expression of 75 consensus LAAs were determined using a custom-designed RT-PCR array. 12 samples underwent extended LAA analysis by flow sorting into “bulk leukemia” and “stem cell enriched” populations. LAA expression was normalized using the geometric mean of three control genes. Results: Samples from 48 AML patients (30 blood, 22 marrow) were suitable for analysis. Average age of patients was 53 (24-86), 50% were female. Cytogenetics were favorable (17%), intermediate (65%) or adverse (19%); 29% presented with a white blood cell count >50,000. Over 10,000 individual data-points were collected. Five distinct patterns of LAA expression were observed in blood from AML patients compared to healthy donors. Conclusions: Understanding the heterogeneity and patterns of AML LAA expression between individuals allows the rational prioritization of potential targets for immunotherapy. Based on our data we predict that targeting any single LAA will likely often be ineffective but that it may be possible to create an inclusive panel of multiple targets with coverage of most AMLs, eliminating the need for individualized personalization of therapy. Such AML antigen signatures may also have utility for minimal residual disease monitoring. [Table: see text]

Abstract 1967 Following allogeneic stem cell transplant (SCT), late-onset severe pulmonary complications occur in more than 10% of patients and confer an increased risk of mortality in the first few years post transplant. Whether pulmonary repair can occur subsequently is not well characterized. We prospectively collected pulmonary data on 138 (60 female, 78 male) recipients of allogeneic SCT from HLA identical siblings surviving > 5 years. The median age at transplant was 36 years. Indications for transplant were CML (63), AML (31), MDS (19), ALL (12) and others (14). 126 patients received ≥1200 cGy TBI-based conditioning with or without lung shielding, 3 received 400 cGy TBI-based conditioning and 9 patients received no irradiation. The graft was ex vivo T cell depleted in 131 recipients except six who received non-myeloablative SCT. Of the survivors, 26 had no cGvHD, 70 had cGvHD lasting less than 3 years and 42 had prolonged cGvHD requiring systemic immunosuppression for more than 3 years. Of the 17 patients who died beyond 5 years, 4 died of pulmonary causes, including two with lung cancer. Pre-transplant abnormalities (< 80% predicted) in PFTs were found in 15.9% of subjects in the following declining frequency: FVC%, VC, TLC, FEV1, DLCO_Adj (adjusted for hemoglobin and alveolar ventilation) Hb/VA and FEV1/FVC. Baseline (BL) PFTs served as the reference for all subsequent measurements at 5, 10 and 15 years for each survivor. The only parameter showing a clinically significant decline post transplant was DLCO_Adj at the 5-year mark, with subsequent normalization [paired t-test]. The median DLCO_Adj at 5, 10 and 15 years were 88%(p Disclosures: No relevant conflicts of interest to declare.

Abstract 2528 The quantitative lymphocyte recovery after remission induction is known to correlate with the outcome of the patients with acute myeloid leukemia (AML), suggesting the immune-surveillance is at least partly responsible for maintained remissions. However defects in lymphocyte function and increased regulatory T cell numbers suggest a permissive immune environment for leukemia cell growth. Understanding the interplay between the leukemia and the immune system may provide insight into the mechanisms governing leukemia cure or relapse after induction chemotherapy and lead to better remission treatments to prevent relapse. To look for evidence of immune escape and possible immunoediting of lymphocyte function by the leukemia, we undertook an extensive phenotypic characterization of lymphocytes and AML blasts in blood and marrow at presentation, through remission to full hematological recovery. Twenty patients (mean age 57 years; range 31–69) with newly diagnosed AML were enrolled in a protocol permitting blood and marrow collection for investigational purposes (VICCHEM 1073). Mononuclear cells were obtained from blood and marrow samples collected at onset, 14–21 days and 28–35 days after start of induction chemotherapy. Mononuclear cells were analyzed for lymphocyte phenotyping by flow cytometry and subset frequencies were compared with 5 healthy controls (mean age 31). Leukemic blast cells (Lineage CD2/3/19 negative, CD45+ cells) were characterized in blood and marrow. T cells showed several, largely persisting, abnormalities: (1) A decrease in central memory CD197+, CD27+, CD45RO+ CD8+ (p=0.03) and CD4+ T cells (p=0.1) in the blood at presentation with a corresponding decrease in CD197+CD27+ CD45RO-naïve subsets (2) A significant increase in PD1+ high expression suppressor phenotype for CD8+ (p=0.001) and CD4+ (p=0.01) cells at recovery (days 28–35). (3) At presentation FoxP3+ CD4+ regulatory T cells (Treg) were increased significantly at presentation (p=0.03) and remained elevated. This increase was due mainly to augmentation in the HeliosloFoxP3hi induced Treg (p=0.04) and CD45RAloFoxP3hi effector Treg fraction (p=0.002). These changes persisted into remission. The leukemic blast population included a variable population of cells with the CD11b+CD124+HLA-DRlo myeloid derived suppressor (MDSC) phenotype. The frequency of MDSC was higher in bone marrow (median 6.1% vs 3.1% in blood; p=0.44) and a significantly higher proportion of marrow MDSC expressed the lymphocyte suppressor molecule PDL-1 (54.5% vs 28.5%; p=0.0189). NK cell subsets showed significantly lower frequencies of immature CD56bright NK cells (p=0.02), and correspondingly decreased more mature CD56dim CD57-KIR-NKG2A+ subsets at presentation (p=0.039). NK cell subsets trend to recover during remission but with reduced frequency of mature CD57+, KIR+ CD56dim NK cells in recovery (p=0.018). This study confirms the occurrence of significant alterations in T cell phenotype associated with increased regulatory and suppressor phenotype. The occurrence of the MDSC phenotype within the leukemia blast population suggests that myeloid leukemia cells promote a permissive immune environment for leukemia growth and persistence through immunoediting. These changes as well as abnormalities in NK cells tend to persist into remission and may contribute to leukemic relapse. Disclosures: No relevant conflicts of interest to declare.

EDITOR—James may be ready for a UK wide system of graduate entry medical education,1 but are we sure that UK medical education is ready for graduates? Absurdities remain at all stages of the current training system—the Joint Committee on Higher Medical Training states that a three year doctorate completed before or during medical …

Both early- and late-onset noninfectious pulmonary injury are important contributors to the nonrelapse mortality seen after allogeneic stem cell transplantation (allo-SCT), particularly in subjects conditioned with high-dose total body irradiation (TBI). To characterize the kinetics of recovery from pulmonary injury in long-term survivors, we collected data on 138 subjects who survived > 3 years (median survival, 10.2 years) after predominantly TBI-based allo-SCT from their HLA-matched siblings. Baseline pulmonary function tests served as the reference for subsequent measurements at 3, 5, 10, and 15 years for each survivor. The only parameter showing a clinically and statistically significant decline post-transplant was adjusted diffusion capacity of lung for carbon monoxide (DLCO), which reached a nadir at 5 years but surprisingly normalized at the 10-year mark. Multivariable modeling identified chronic graft-versus-host disease (P < .02) and abnormal baseline-adjusted DLCO (P < .03) as the only significant factors associated with the decline in adjusted DLCO at 5 years but excluded smoking, conditioning intensity, baseline C-reactive protein level, TBI dose to the lungs, disease, and demographic variables. In conclusion, pulmonary injury as monitored by the adjusted DLCO continues to deteriorate in the first 5 years after allo-SCT but recovers at 10 years.

We report a case of t(8;21) acute myeloid leukemia presenting as severe aplastic anemia. While initial bone marrow biopsy lacked any cytogenetic abnormalities in 20 analyzed metaphases, repeat bone marrow biopsy eight days later demonstrated this translocation. Initial cytogenetic analysis of 20 metaphases was therefore insufficient to make the diagnosis of hypocellular acute myeloid leukemia. We discuss that further complementary molecular tests, such as CGH, would likely provide a more robust diagnosis of hematopoietic diseases., Highlights • It is difficult to distinguish between hypocellular MDS, hypocellular AML and SAA. • Acute leukemia may present as SAA. • 20 Metaphases may be insufficient to diagnose hypocellular AML. • Complementary methods, such as SNP based CGH arrays, would enhance the diagnosis.