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Background: Although some studies have validated the 2001 WHO classification of acute myeloid leukemia (AML), including the importance of multilineage dysplasia, others have suggested that multilineage dysplasia correlates with unfavorable cytogenetics but has no independent impact on prognosis. In 2008, the revised WHO classification system has expanded this category into “AML with myelodysplasia-related changes” (AML-MRC) that now includes 1) AML arising from myelodysplastic syndrome (MDS), 2) AML with MDS-related cytogenetic abnormalities, and 3) AML with multilineage dysplasia. An individual case may fall into this category by meeting any of the criteria. The goal of the current study is to clinically characterize this newly defined AML-MRC subgroup. Methods: One-hundred consecutive AML patients diagnosed at Stanford University Hospital between 2005 and 2007 with adequate material for mutation analysis were studied. Cases were classified using the 2008 WHO criteria. Diagnostic cytogenetic findings were reviewed and patients were stratified into risk groups using Southwest Oncology Group criteria. Available flow cytometry immunophenotyping results were reviewed and all samples were tested for NPM, FLT3 (ITD and D835) and CEBPA mutations. Clinical parameters including hemogram data at time of diagnosis were reviewed. Clinical follow-up including overall survival (OS), progression free survival (PFS) and complete remission (CR) rates were retrospectively determined. Kaplan-Meier methods and univariate and multivariate Cox proportional hazards regression analysis were used to compare the clinical data. Results: The cases included 57 males and 43 females with a median age of 56 (range 17–81). Cytogenetic risk-group stratification resulted in 9 patients with favorable, 65 with intermediate and 19 with unfavorable risk status. Using the 2008 WHO criteria, there were 48 AML-MRC, 40 AML not otherwise specified (AML-NOS), 9 AML with either t(8;21), inv(16) or t(15;17), and 3 therapy related AMLs. Overall, 26 patients had a NPM1 mutation (16 of which were FLT3 mutated), 25 had FLT3-ITD, 8 had FLT3-D835 and 9 had a CEBPA mutation (3 of which were FLT3 mutated). Compared to AML-NOS, patients with AML-MRC were significantly older (59 vs 51 years, p=0.014) and presented with lower hemoglobin (9 vs 11.2 g/dL, p=0.044), lower platelets (47 vs 54 K/uL, p=0.059), unfavorable cytogenetics (14/46 vs 3/36, p=0.014) and exhibited a decreased frequency of CEBPA mutation (0/46 vs 7/40, p=0.001) as compared to AML-NOS. Based on the flow cytometry immunophenotyping, the blasts from patients with AML-MRC more frequently expressed CD14 compared to AML-NOS (10/46 vs 4/36, p=0.048). Clinical outcome data showed that patients with AML-MRC had a significantly worse OS, PFS and CR compared to AML-NOS (Figure, all p 60), FLT3-ITD and AML-MRC status as significant predictors of worse OS with the following respective hazard ratios: 2.82 (95% CI, 1.52–5.26), 2.11 (1.01–4.42), 1.98 (1.01–3.90), 1.92 (1.01–3.65). Conclusion: The newly defined WHO category of AML-MRC exhibits a significantly worse clinical outcome compared to AML-NOS and is predictive of worse overall survival in the multivariate analysis of AML patients, independent of age or cytogenetic risk group. These findings support the clinical, morphologic and cytogenetic criteria for this 2008 WHO AML category. Figure Figure

Dysregulated Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F–negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis.

Abstract 2863 Background: JAK2 V617F is a recurrent, activating mutation in patients (pts) with BCR-ABL1-negative MPNs. Mutations in codon 515 of MPL occur in 1–5% and 5–10% of ET and PMF pts, respectively, and similar to JAK2 V617F, lead to constitutive JAK-STAT signaling. The acquisition of multiple mutational events affecting the JAK-STAT axis or components of the epigenetic machinery is common in MPNs and likely contributes to phenotypic diversity, including progression to acute myeloid leukemia. Mutated genes thus far implicated in MPN initiation and/or progression include TET2, CBL, SH2B3, ASXL1, DNMT3A, IDH1/2, IKZF1, EZH2, SRSF2, and TP53. In order to identify novel somatic mutations associated with classic BCR-ABL1-negative MPNs, we performed whole genome sequencing of DNA extracted from peripheral blood granulocytes and cultured skin fibroblasts from a patient with PMF and a known MPL W515K mutation. Methods: Whole genome sequencing (WGS) was undertaken in a 55 year-old man with untreated PMF four years after initial diagnosis. His DIPSS Plus risk group was low (score 0). His karyotype was normal, and molecular testing revealed wild-type JAK2 in addition to the MPL W515K mutation. WGS of purified granulocytes and paired cultured skin fibroblasts was performed using both Illumina HiSeq and Complete Genomics (CGI) platforms. The resulting data were analyzed using multiple independent aligners and variant callers. Amplicon-based targeted resequencing with the Illumina MiSeq platform was used to evaluate additional patient samples for recurrent mutations. Stanford institutional review board approval and informed pt consent was obtained for these analyses. Results: The PMF genome was sequenced to 88X (Illumina) and 128X (CGI) average fold coverage, and the cultured skin fibroblast genome was sequenced to 47X (Illumina) and 126X (CGI). The PMF genome had a low somatic mutation rate, consistent with that observed for other sequenced hematopoietic tumor genomes, with a low number of true somatic mutation calls. To definitively identify true mutations among various sequencing artefacts and germline variants, we use cultured skin fibroblasts which can be prepared with no contamination by neoplastic cells. In addition to re-identification of the MPL W515K mutation, this approach identified six additional somatic mutations that alter gene coding regions, splice sites, or known regulatory regions: a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; a splice-site mutation in CAP2; three nonsynonymous point mutations in KIAA0355, SOX30, MFRP; and a 19-base pair (bp) deletion involving a regulatory region in the 5'-untranslated region (5'-UTR) of BRD2, a bromodomain-containing protein implicated in transcriptional regulation (Table). CARD6, BRD2, and KIAA0355, an uncharacterized protein, are expressed by the granulocytes derived from this patient, supporting a potential role in the development of PMF in this pt. Using massively parallel sequencing, we are currently examining the transcribed region of BRD2 and the coding region of the other five genes in additional pt samples. Analysis of the first 87 samples (MF=47, PV=20, ET=20) out of a cohort of 180 MPN pts has thus far not identified recurrent somatic mutations in these six genes. Conclusion: High-coverage genome sequencing of neoplastic and germline cells from a patient with MPL-mutated PMF identified six additional somatic mutations of potential functional significance. Work is ongoing to determine if somatic mutations in these genes are found in other patients with BCR-ABL1-negative MPNs, and their pathogenetic relevance to MPN biology. Disclosures: Snyder: Illumina: Consultancy; GenapSys: Membership on an entity's Board of Directors or advisory committees; Personalis: Consultancy, Founder Other.

New high-throughput DNA sequencing methods have many advantages for the analysis of rearranged immunoglobulin (Ig) and T cell receptor (TCR) loci in suspected lymphoid malignancies. Instead of primarily analyzing the size distribution of rearranged immune receptors using gel or capillary electrophoresis, sequencing complex nucleic acid pools provides much more complete characterization of the receptor sequences present. Currently, separate assays are used to evaluate Ig and TCR clonality, Ig hypermutation status, and to test for residual malignant cells after treatment. We used multiplexed PCR amplification of immunoglobulin heavy chain loci for high-throughput pyrosequencing with the Roche 454 platform to evaluate the detection of clonal IgH sequences, determination of hypermutation status, and sensitivity of detection of low levels of a known clonal receptor. Our method also evaluated the feasibility of physical “bar-coding” of DNA molecules derived from different samples to enable cost-effective sequencing of sample pools. 23 pooled specimens were sequenced including: known lymphoma specimens (4); peripheral blood leukocytes from normal individuals (4); specimens with indeterminate or discordant results by conventional clonality assays (7); and a dilution series of a clonal CLL/SLL specimen into normal peripheral blood leukocytes. Our initial experiment characterized 522,099 IgH sequences from these specimens, with a mean read length of 242 base pairs. We have employed IgBlast (NCBI) for initial alignment of rearranged IgH sequences to germline V, D and J gene segments. Our data demonstrate that clonal IgH populations can be readily detected and characterized as to their germline or hypermutated status and unique V-D and D-J junctions with this approach. In our dilution series experiment, the clonal receptor sequence could be reliably observed after 1000-fold dilution. Normal patient specimens show expected extensive sequence diversity, and we are exploring methods for optimally summarizing and representing these data. Some PCR artifacts, as well as non-immunoglobulin sequences commonly amplified by current IgH primer sets were also detected, and suggest avenues for further improvement of the methods used for detection and characterization of clonal immune receptors. More broadly, these methods also offer a novel approach to monitoring normal immune responses such as the response of patients to vaccination, and of characterizing the immune system in patients with autoimmune diseases.

Following liver transplantation, patients require immunosuppressive medications to prevent graft rejection. However, these drugs can have significant side effects including severe anemia. Tacrolimus is now commonly used in pediatrics patients after liver transplantation. Case reports indicate that tacrolimus rarely can cause microangiopathic hemolytic anemia and also hemolytic anemia due to cold reactive IgM antibodies (cold agglutinin disease). We have seen 4 patients with symptomatic cold agglutinins thought to have been triggered by tacrolimus. Four patients, 6 to 26 months post-ABO-compatible liver transplant, presented with severe anemia (hemoglobin < 6g/dL), indirect hyperbilirubinemia, reticulocytosis and low serum haptoglobin (Table 1). Peripheral blood smear exhibited marked autoagglutination but no evidence of microangiopathic changes. The direct antiglobulin test (DAT) initially was strongly positive in all 4 patients (Table 2). After warm saline washes, the DAT remained positive for complements in 3 out of 4 patients. Cold agglutinin titers were low (1:4 – 1:16) but thermal amplitude studies were positive at 37°C in the two tested patients. Viral serologies significant for cold agglutinin syndrome were negative. None of the patients had clinical evidence of post-transplant lymphoproliferative disorder. All 4 patients had brisk hemolysis requiring multiple uncrossmatched packed red blood cells (PRBC) transfusions. Favorable resolution of the hemolytic anemia occurred following steroids, plasmapheresis and withdrawal of tacrolimus. Subsequently, patients were placed on cyclosporin and none exhibited liver rejection or recurrence of their hemolytic anemia. The above observation indicates that acute cold-agglutinin induced hemolysis occurs in patients following liver transplantation and these events appear to have temporally associated with tacrolimus administration. Patient Profiles Patient Age Diagnosis Months after transplant Presenting hemoglobin (g/dL) Reticulocyte (%/Abs k/μL Total/Direct Bilirubin Treatments (in addition to withdrawal of tacrolimus) ND=not done. MP=methylprednisolone 1 8 mos Neonatal iron storage disease 6 2.8 41%/469 4.2/0.6 MP 4mg/kg/d plus 30mg/kg/d x 3 days. Plasmapheresis x 6. 2 13 mos Biliary atresia Idiopathic 6 5.3 8%/250 3.2/0.4 MP 4mg/kg/d. Plasmapheresis x 15. 3 2 yrs 5 mos fulminant hepatic failure 14 4.6 19.5%/237 2.6/0.3 MP 10mg/kg/d. Plasmapheresis x 10. 4 4 yrs Biliary atresia 26 5.3 5%/ND 8.8/0.5 MP 2mg/kg/d→ 4mg/kg/d. Plasmapheresis x 10. Immunohematology Profiles Admission DAT Patient 1 Patient 2 Patient 3 Patient 4 Admission DAT Pre/post saline wash Pre/post saline wash Pre/post saline wash Pre/post saline wash DAT-Direct antiglobulin test, M-microscopically, NT-not tested, +-positive, 0-negative, Polyspecific 3+/0 1+/M+ 4+/4+ 3+/1+ Anti-IgG 2+/0 M+/0 4+/1+ 3+/1+ Anti-C3 2+/0 2+/1+ 4+/4+ 3+/1+ Saline Control 2+/0 1+/0 1+/0 1+/0 Eluate 0 NT Panreactive 1+ Panreactive 1+ Cold agglutinin titer at 4°C in saline NT 4 8 16 Thermal amplitude screen at 30°C NT NT Reactive Reactive Donath-Landsteiner Test NT NT Negative Negative Admission peripheral blood smear 4+ polychromatophilia. 4+ microcytosis 4+ autoagglutination. 3+ polychromatophilia. 3+ spherocytes. 3+ microcytes 3+ autoagglutination. 3+ polychromatophilia. 3+ spherocytes. 2+ macrocytes 4+ autoagglutination. 4+ spherocytes. 2+ polychromatophilia. 2+ macrocytes