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Background: Myeloid neoplasms and reactive conditions cannot be readily distinguished by conventional CBC parameters. Given the time, expense and clinical expertise associated with the diagnostic workup of myeloid neoplasms, there is a clear need for a cost-effective screening laboratory test that can rapidly and accurately select for patients with cytopenias or cytoses potentially related to myelodysplastic syndrome (MDS) or myeloproliferative neoplasms (MPN), respectively. Recent studies showed that incorporating NE-WX, a research CBC parameter related to neutrophil complexity, and IPF, an immature platelet fraction parameter, into a conventional CBC analysis helps identify patients with high likelihood of MDS (PMID 30406952) or JAK2-positive polycythemia vera (PMID 30601597), respectively. Our exploratory analysis was focused on using all research CBC parameters in combination with standard CBC parameters and mutational data to compare control patients to those with active myeloid disease, and MDS in particular. Methods: We measured standard and research CBC parameters (92 parameters overall) using the Sysmex XN series hematology analyzer in a cohort of 592 consecutive patients (pts), out of which 221 had abnormal CBC due to an underlying myeloid neoplasm (MDS 83 pts, MPN 54 pts, MDS/MPN 24 pts, acute myeloid leukemia 21 pts, clonal or non-clonal cytopenia or cytoses 33 and 6 pts, respectively) and 371 age-matched control patients with normal CBC. The former group was tested for the presence of somatic mutations in 95 genes associated with hematologic malignancies and included patients with active disease (n=127) and in remission (n=94). The MDS cohort included 59 patients with active disease and 24 patients in remission. We developed a variety of models (Linear and Logistic Regression, Decision Trees, Random Forests, Neural Networks, Support Vector Machines, Gradient Boosting) to predict the presence of an active myeloid neoplasm or active MDS using both CBC and molecular data, as well as CBC data alone. These models were compared using a leave one patient out cross-validation method and a champion model was chosen. Correlation matrix analysis was used for establishing relationships between MDS-associated mutations and CBC parameters, where a relevant association was considered at the levels of above 0.25 or below -0.25. Results: Table 1 lists the leave one out performance of the champion predictive models for the active myeloid and MDS cohorts based on different variables (all CBC parameters, mutations, age and gender), along with their corresponding sensitivity and specificity. Table 2 shows the most discriminatory research CBC parameters in the MDS cohort reflecting dysplastic changes in neutrophil morphology (NE-SFL, NE-WY, NE-WX, NE-FSC, NE-SSC) and degrees of cytopenia (increased IPF and decreased TNC). The standard parameters included Hgb, MCV, MCH, WBC and absolute neutrophil count. Several mutations showed a strong association with certain CBC parameters in the MDS cohort. ASXL1 was associated with lymphocyte complexity (LY-X; 0.38), TNC (0.28), WBC (0.28) and neutrophil (0.27) count and lower immature reticulocytes fraction (-0.67), RBC (-0.30) and reticulocyte (-0.31) counts, similar to the findings of the recently published mouse model expressing ASXL1 truncating protein (PMID 29113963), while DNMT3A and SF3B1 mutations showed higher platelet correlations (0.32). In addition, the latter was associated with lower neutrophil granularity (-0.32), cell size (-0.32) and nuclear complexity (-0.39). TP53 showed positive association with neutrophil and lymphocyte parameters reflecting population heterogeneity (0.26-0.40). Conclusions: Our exploratory study demonstrated that the Sysmex XN research parameters, which are measured with all routine CBCs, may be used in patients with abnormal CBC to build a predictive model with high specificity to rule out a clonal myeloid neoplasm, including MDS. The model's performance improves with the addition of the molecular status as a variable, while it appears to be independent of age and gender. In the entire MDS cohort, the most relevant research parameters in the predictive model are reflective of dysplastic neutrophil morphology and can be objectively measured. In addition, it appears that there may be unique CBC parameter signatures associated with an underlying mutation. Disclosures Pozdnyakova: Sysmex corporation of America: Research Funding. Niculescu:Sysmex America, Inc: Consultancy. Kroll:Sysmex America, Inc: Consultancy. Kim:Quanterix, Inc: Consultancy; Papgene, Inc: Consultancy; LabCorp, Inc: Consultancy.

This interactive case features a 52-year-old man of West African origin who presents with nausea, vomiting, and profound lethargy. Test your diagnostic and therapeutic skills at NEJM.org.

Personalized medicine requires physicians to employ complex and expensive diagnostic analyses. However, without evidence-based standards, there is over-utilization of testing that complicates patient care, diminishes quality, and increases costs. To address this, we implemented the diagnostic management team (DMT), a multi-disciplinary system for development and deployment of diagnostic testing guidelines for hematologic malignancies. The team created evidence-based standard ordering protocols (SOPs) for cytogenetic and molecular testing that were applied by pathologists to bone marrow biopsies on adult patients. Testing on 780 biopsies performed during the 6 months before SOP implementation was compared with 1,806 biopsies performed during the subsequent 12 months. After implementation, there were significant decreases in tests discordant with SOPs, omitted tests, and the estimated cost of testing to payers. The fraction of positive tests increased. Clinicians reported acceptance of the new procedures and perceived time savings. This process is a model for optimizing complex and personalized diagnostic testing.

Hemophagocytic lymphohistiocytosis (HLH) is a frequently fatal and likely underdiagnosed disease involving a final common pathway of hypercytokinemia, which can result in end-organ damage and death. Although an early diagnosis is crucial to decrease mortality, the definitive diagnosis is often challenging because of the lack of specificity of currently accepted diagnostic criteria and the absence of confirmatory gold standards. Because of the wide range of laboratory assays involved in the diagnosis of HLH, practicing pathologists from a broad spectrum of clinical specialties need to be aware of the disease so that they may appropriately flag results and convey them to their clinical counterparts. Our article summarizes these new advances in the diagnosis of HLH and includes a review of clinical findings, updated understanding of the pathogenesis, and promising new testing methods.

Measurement of minimal residual disease is routine in diseases such as chronic myelogenous leukemia, precursor B cell acute lymphoblastic leukemia, and acute promyelocytic leukemia because it provides important prognostic information. However, the role of minimal residual disease testing has not been widely adopted in multiple myeloma (MM), with other parameters such as the International Staging System (ISS) and cytogenetic analysis primarily guiding therapy and determination of prognosis. Until recently, achieving a complete response (CR), as defined by the International Myeloma Working Group (IMWG) criteria, was rare in patients with MM. The use of novel agents with or without autologous peripheral blood stem cell transplantation (auto-PBSCT) has significantly increased CR rates, thus increasing overall survival (OS) rates. The majority of patients with MM have persistent levels of residual disease that are below the sensitivity of bone marrow (BM) morphology, protein electrophoresis with immunofixation, and light chain quantitation even after attaining CR and will eventually relapse. Measurement of minimal residual disease by more sensitive methods, and the use of these methods as a tool for predicting patient outcomes and guiding therapeutic decisions, has thus become more relevant. Methods available for monitoring minimal residual disease in MM include PCR and multiparameter flow cytometry (MFC), both of which have been shown to be valuable in other hematologic malignancies; however, neither has become a standard of care in MM. Here, we review current evidence for using minimal residual disease measurement for risk assessment in MM as well as incorporating pretreatment factors and posttreatment minimal residual disease monitoring as a prognostic tool for therapeutic decisions, and we outline challenges to developing uniform criteria for minimal residual disease monitoring.

A dogma in squamous epithelial biology is that proliferation occurs in the basal cell layer. Notable exceptions are squamous epithelia of the human oral cavity, esophagus, ectocervix, and vagina. In these human epithelia, proliferation is rare in the basal cell layer, and the vast majority of cells positive for Ki67 and other proliferation markers are found in para- and suprabasal cell layers. This unique human feature of a generally quiescent basal cell layer overlaid by highly proliferative cells offers the rare opportunity to study the molecular features of undifferentiated, quiescent, putative stem cells in their natural context. Here, we show that the quiescent human oral mucosa basal cell layer expresses putative markers of stemness, while para- and suprabasal cells are characterized by cell cycle genes. We identified a TGFβ signature in this quiescent basal cell layer. In in vitro organotypic cultures, human keratinocytes could be induced to express markers of these quiescent basal cells when TGFβ signaling is activated. The study suggests that the separation of basal cell layer and proliferation in human oral mucosa may function to accommodate high proliferation rates and the protection of a quiescent reserve stem cell pool. Psoriasis, an epidermal inflammatory hyperproliferative disease, exhibits features of a quiescent basal cell layer mimicking normal oral mucosa. Our data indicate that structural changes in the organization of epithelial proliferation could contribute to longevity and carcinogenesis.

Pediatric follicular lymphoma shares morphologic similarities with the adult form of the disease but lacks other classic features of adult lymphoma, including t(14;18) translocation, BCL2 overexpression, and transformation to aggressive higher-grade lymphoma. Herein, we report a novel case in which a 5-year-old boy (ethnicity unknown) had follicular lymphoma, along with concurrent high-grade and clonally related disease that fulfilled all of the morphologic, immunophenotypic, and genetic criteria for Burkitt lymphoma, including a t(8;14) translocation involving the MYC gene. To our knowledge, this case is the first reported instance of transformation of follicular lymphoma of any sort into true Burkitt lymphoma and the first reported instance of acquisition of MYC abnormalities in pediatric follicular lymphoma.

Viruses are leading causes of severe acute lower respiratory infections (LRIs). These infections evoke incomplete immunity, as individuals can be repeatedly reinfected throughout life. We report that acute viral LRI causes rapid pulmonary CD8+ cytotoxic T lymphocyte (TCD8) functional impairment via programmed death-1/programmed death ligand-1 (PD-1/PD-L1) signaling, a pathway previously associated with prolonged antigenic stimulation during chronic infections and cancer. PD-1-mediated TCD8 impairment occurred acutely in mice following infection with human metapneumovirus or influenza virus. Viral antigen was sufficient for PD-1 upregulation, but induction of PD-L1 was required for impairment. During secondary viral infection or epitope-only challenge, memory TCD8 rapidly reexpressed PD-1 and exhibited severe functional impairment. Inhibition of PD-1 signaling using monoclonal antibody blockade prevented TCD8 impairment, reduced viral titers during primary infection, and enhanced protection of immunized mice against challenge infection. Additionally, PD-1 and PD-L1 were upregulated in the lungs of patients with 2009 H1N1 influenza virus, respiratory syncytial virus, or parainfluenza virus infection. These results indicate that PD-1 mediates TCD8 functional impairment during acute viral infection and may contribute to recurrent viral LRIs. Therefore, the PD-1/PD-L1 pathway may represent a therapeutic target in the treatment of respiratory viruses.

AimsHaemophagocytic lymphohistiocytosis (HLH) is divided into paediatric (primary) and adult (secondary) types. While paediatric-HLH has been extensively characterised, similar studies in adults are limited. This study aims to evaluate the significance of the HLH diagnostic criteria as well as other clinical parameters in adults with bone marrow evidence of haemophagocytosis.MethodsWe conducted a 10-year retrospective search of the pathology archives of two institutions for cases with bone marrow haemophagocytosis. We included those cases that fulfilled the currently established HLH diagnostic criteria. For the 29 cases that met inclusion criteria, we assessed clinical features, co-morbidities, therapy and clinical outcome. The effect of 19 clinical variables on mortality outcomes was assessed using logistic and hazard regression analyses.ResultsOf cases for which an aetiology could be identified, infectious diseases were the most common association (14 of 19, 74%). Fever and elevated ferritin were the most frequently available criteria used to establish HLH. The overall mortality rate was 61% despite HLH-specific therapy, which had been initiated in 48% of the cases. The remaining cases were treated with supportive therapy and antibiotics. The most statistically significant marker of mortality was an elevated absolute neutrophil count (ANC), a feature not typical of HLH.ConclusionsSince elevated ANC correlates with poor outcomes in sepsis, and not HLH, we postulate that many of the patients fulfilling HLH diagnostic criteria in this study likely had sepsis/systemic inflammatory response syndrome rather than HLH. Our results highlight the need to define HLH diagnostic criteria specific to the adult population.

Gene fusions involving the catalytic domain of tyrosine kinases (TKs) are found in a variety of hematological and solid tumor malignancies. Clinically, TK fusions have emerged as prime targets for therapy with small molecule kinase inhibitors. Unfortunately, identification of TK fusions has been hampered by experimental limitations. Here, we developed version 2.0 of a genomically based systematic kinase fusion screen and used it to detect a novel imatinib-sensitive C6orf204-PDGFRB fusion in a patient with precursor T lymphoblastic lymphoma (T-ALL) and an associated myeloproliferative neoplasm with eosinophilia. These data validate the ability of this targeted capture-sequencing approach to detect TK fusion events in small amounts of DNA extracted directly from patient samples.

A marked peripheral blood eosinophilia is an uncommon finding in a complete blood count (CBC). According to Wardlaw and Kay (Eosinophils and Their Disorders. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn U, editors. Williams Hematology. 6(th) ed. New York: McGraw-Hill, 2001. p. 790-93), the most common causes are infection by helminthic parasites, atopic disease, and, less commonly, primary hypereosinophilic syndromes. Therefore, when eosinophilia is seen in a CBC, it can provide an important clue to the correct diagnosis. We present a case of a patient with a finding of pulmonary nodules in the setting of cancer and a CBC finding of profound peripheral blood eosinophilia. As a result of the high level of clinical suspicion for Coccidioides infection due in part to the eosinophilia, adequate steps were taken in the clinical laboratory not only to correctly diagnosis the patient, but also to protect the laboratory staff from work-related exposure to this easily aerosolizable infectious agent.

Background: Recent studies (Coombs et al., Cell Stem Cell 2017) have identified presence of clonal hematopoiesis of indeterminate potential (CHIP) in samples of solid tumors. CHIP is more prevalent among cancer survivors who subsequently develop therapy-related myeloid neoplasm (Gillis et al., Lancet Oncol 2017; Takahashi et al., Lancet Oncol 2017; Gibson et al., J Clin Oncol 2017). However, the relationship between CHIP and exposure to myelotoxic chemotherapy delivered as part of treatment for solid tumor is uncertain. We hypothesized that CHIP is more prevalent among recipients of myelotoxic chemotherapy compared with age-matched population. Methods: In this prospective, cross-sectional study, we collected peripheral blood samples from survivors of breast cancer or lymphoma who had received anthracycline- and/or alkylator-containing chemotherapy as part of their curative cancer therapy. All subjects had to be clinically free of cancer, and not have any hematologic disorders or unexplained cytopenias. We recruited patients age 50 to 70, because according to published population datasets (Jaiswal et al. and Genovese et al., NEJM 2014) in a cohort with mean age of 60 the expected CHIP prevalence would be about 5%. To minimize any potential contamination by circulating tumor cells, we isolated genomic DNA from purified CD45+ cells. We determined presence of CHIP by next-generation sequencing using an Illumina TruSeq Custom Amplicon kit (MiSeq V2.2). The assay targeted 757 coding exons of 96 genes commonly mutated in hematologic malignancies, including 20 CHIP-defining genes. To establish presence of CHIP, we required a known pathogenic variant with variant allele fraction (VAF) ≥ 2%. According to a pre-specified statistical plan, assuming one-sided alpha of 0.05, the study had 80% power to reject the null hypothesis of baseline CHIP prevalence of 5% in a cohort with sample size of 80. Results: Among 80 enrolled subjects, median age was 62 years (interquartile range, 56-67). There were 78% women, and 88% of subjects were white non-Hispanic. Patients had received doxorubicin- and/or cyclophosphamide-containing adjuvant or curative chemotherapy for breast cancer (56%) or lymphoma (44%). Median time from completion of chemotherapy to enrollment was 27 months (interquartile range, 11-59). We have completed sequencing of 72 samples (updated analysis will be provided at the meeting). Mean coverage depth was 1418x (±224), and ≥200x coverage was achieved in a mean 91.4% (±1.8%) of target amplicons. We detected CHIP in 12 subjects (17%; binomial 95% confidence interval: 10-27%; P=.0002 for the null hypothesis test of 5% prevalence). Mean VAF for the CHIP mutations was 5.3% (range, 1.4% to 29.9%), and patients had up to 4 CHIP-associated mutations (Fig. A). The CHIP-associated mutations had a typical distribution with most common mutations in DNMT3A, ASXL1, SRSF2, and TET2 (Fig. B). There was only 1 TP53 mutation, previously suggested to associate with exposure to chemotherapy (Coombs et al., 2017). Potentially germline variants of unknown significance (VUS) were found in 78% of patients, at mean VAF 49% (Fig. C), most commonly in ATM (12%), NOTCH2 (7%), BCORL1, and DNMT3B (6% each). Additionally, 2 patients had low-VAF variants suspicious for CHIP: ATM c.6059G>A (3.0%); PRPF8 c.790T>C (VAF 2.3%). Presence of CHIP was not significantly associated with age (within the narrow age range in the study cohort), sex, race, type of cancer (breast or lymphoma), count of white cells, red cells, platelets, or time elapsed from completion of chemotherapy. Conclusions: We have detected a significantly increased, more than 3 times the expected value, prevalence of CHIP among cancer survivors who had received myelotoxic chemotherapy. However, the distribution of mutations was typical for CHIP, without previously suggested over-representation of TP53. Further research is ongoing to determine whether presence of CHIP is related to a direct mutagenic effect of chemotherapy or competitive advantage of pre-existing CHIP clones after the hematopoietic stress of chemotherapy. Our data indicate that an affordable next-generation sequencing screen may be useful for detection of CHIP in cancer patients who are planning adjuvant chemotherapy, or as a surveillance tool after such therapy, to predict the risk of a therapy-related myeloid neoplasm and optimize personalized treatment strategies. Disclosures Olszewski: TG Therapeutics: Research Funding; Genentech: Research Funding; Spectrum Pharmaceuticals: Consultancy, Research Funding. Kim:Aushon Biosciences: Consultancy; LabCorp, Inc.: Consultancy; Papgene, Inc: Consultancy. Fenton:Astellas Pharma US: Other: Spouse employment. Reagan:Alexion: Honoraria; Takeda Oncology: Research Funding; Pfizer: Research Funding.

Background With the increasing accessibility of next-generation sequencing (NGS), many institutions are incorporating routine mutational profiling to assist in the evaluation of patients with a variety of hematologic disorders, including cytopenias. Such testing can be helpful in distinguishing neoplastic causes of cytopenias from others. Mutation profiling of peripheral blood is a particularly attractive option because of its potential application as a minimally invasive screen for hematologic malignancies, particularly myeloid neoplasms. However, it is not entirely clear how such molecular data can inform hematology practice as there is limited data on the clinical value of routine NGS testing in cytopenic patients. Here we report the clinical utility of peripheral blood screening by targeted NGS testing in a large institutional cohort of patients with cytopenias. Methods After institutional review board approval, we identified all patients presenting with peripheral blood cytopenias over a 30-month period (January 2015 - June 2017) to the Adult Hematology Clinic at Dana-Farber/Brigham and Women's Cancer Center (n=1491). Of these, 244 patients (median age: 66, range: 22-93) underwent testing of a peripheral blood specimen using a custom 95-gene, amplicon-based sequencing panel (Rapid Heme Panel, Kluk et al., 2016) surveying genes recurrently mutated in hematologic malignancies (Table 1). In addition, these patients also received a complete hematologic work-up to determine the cause for the cytopenia(s). All patients with a known history of a hematologic malignancy were excluded. Results Overall, 60 (25%) patients had a pathogenic somatic mutation in at least one of the 95 genes studied (Figure 1). An underlying hematologic malignancy was identified in 26 (44%) of these patients at the time of presentation, most commonly a myeloid neoplasm (19/26; 73%); 11 (18%) had a non-neoplastic etiology of the cytopenia(s); and 23 (38%) had unexplained cytopenia(s) following complete work-up. The most frequently mutated genes in patients with unexplained cytopenias were TET2, SF3B1 and SRSF2. Of the 184 (75%) patients without a pathogenic somatic mutation, 15 (8%) of these patients were found to have an underlying hematologic malignancy at the time of presentation, most commonly a lymphoid neoplasm (11/15; 73%); 84 (46%) had a non-neoplastic etiology of the cytopenia(s); and 85 (46%) had unexplained cytopenia(s) following complete work-up. With a median follow-up of 22 months, two patients with non-clonal unexplained cytopenias went on to develop MDS and splenic marginal zone lymphoma, respectively. Overall, the presence of a pathogenic mutation was strongly associated with the diagnosis of a myeloid neoplasm (RR 11.6, 95% CI 4.5-29.8). Conversely, if no pathogenic mutation were identified, the likelihood of developing a myeloid neoplasm in 2 years was low (5/184; 2.7%). To further investigate the diagnostic utility of peripheral blood mutational profiling as a screening test in predicting the presence of a myeloid neoplasm on bone marrow biopsy, we studied its diagnostic characteristics using bone marrow biopsy diagnosis within a 6-month interval as the gold standard (n=57). The absence of a pathogenic mutation in peripheral blood was highly predictive of absence of a myeloid neoplasm on bone marrow biopsy (NPV 94%, 95% CI 84-99%). The presence of specific mutations in spliceosome genes (SF3B1, SRSF2, U2AF1) and co-mutation patterns involving DNMT3A, TET2 and ASXL1 genes and other genes were predictive of a myeloid neoplasm with positive predictive values of 75% (95% CI 41-93%) and 70% (95% CI 41-88%) respectively. Finally, larger clone sizes (≥20%) and higher numbers of mutations (≥2) were also predictive of a myeloid neoplasm (PPV: 80%, 95% CI 60-90% and 75%, 95% CI 49-90%, respectively). Conclusions In conclusion, mutation profiling of peripheral blood is a valuable minimally invasive test in the diagnostic work-up of the cytopenic patient. After a complete hematologic work-up and in the absence of a pathogenic somatic mutation, the likelihood of developing a myeloid neoplasm in 2 years is very low. Also, the lack of a pathogenic mutation in peripheral blood is highly predictive of the absence of a myeloid neoplasm on a bone marrow biopsy. Finally, certain molecular features (number and type of mutations, clone size) are predictive of a myeloid neoplasm. Disclosures Kim: LabCorp, Inc.: Consultancy; Papgene, Inc: Consultancy; Aushon Biosciences: Consultancy.

Background: Immunologic therapies targeting lineage-specific antigens such as CD22 and CD19 can improve outcome in patients with B cell acute lymphoblastic leukemia (B-ALL). The presence of mixed lineage leukemia (MLL) gene rearrangements highlights a subset of B-ALL patients with increased risk of treatment failure after inotuzumab ozogamicin (INO), an anti-CD22 antibody-drug conjugate. In some MLL-rearranged cases, a switch of hematopoietic lineage from B-ALL to acute myeloid leukemia (AML), has been associated with resistance to lymphoid-targeted therapies. In AML, founder clones harboring mutations associated with clonal hematopoiesis of indeterminate potential (CHIP) can persist during remission as reservoirs of treatment resistance. We therefore hypothesized that B-ALL can arise in the setting of a founding CHIP clone and undergo lineage switch under the selection pressure of B cell restricted therapy. Case Description: A 65-year-old female presented with two weeks of easy bruising, fatigue, and 54% circulating blasts. Flow cytometry was consistent with B-ALL that was positive for CD19, CD22, and aberrant CD15. Cytogenetics confirmed the presence of a t(4;11) (q21;q23) MLL rearrangement. The patient received intensive induction with dose-adjusted Larson regimen and morphologic complete remission was confirmed on Day 36. No residual disease was detectable by multiparametric flow cytometry. During consolidation (Day 136), a relapse of CD19+ CD22+ B-ALL was observed in the blood and marrow and treatment with INO was initiated. Despite rapid initial clearance of circulating disease, peripheral blasts were again observed on Day 167. At this time, flow cytometry demonstrated myeloblasts (positive for CD13, CD33, and CD117, but negative for CD19 and CD22), with no evidence of the previous lymphoblast population. The AML was refractory to multiagent salvage chemotherapy. Results: We evaluated the lineage-specificity and clonal dynamics of somatic genetic alterations using targeted gene panel sequencing at 5 timepoints during the course of treatment, compared to results from cytogenetic analysis and flow cytometry. At the time of initial diagnostic evaluation, we identified somatic mutations in DNMT3A (p.Q696*) and TET2 (p.L1209P). Variant allele fractions (VAFs) were 44% and 48%, respectively, suggesting they were both present at high abundance in the leukemic clone. TET2 and DNMT3A mutations are most commonly found in individuals with myeloid neoplasms and CHIP and are not frequently observed in those with B-ALL. We therefore asked whether these mutations preceded the MLL rearrangement in the leukemia clonal hierarchy by comparing their abundance at diagnosis to their abundance during MRD-negative clinical remission. We observed persistence of the DNMT3A (VAF = 13% and 10%) and TET2 (VAF = 8% and 6%) mutations during remission (Day 39, Day 82), while the MLL rearrangement was undetectable at both remission timepoints. We next evaluated the clonal relationship between the myeloid relapse after INO treatment and the initial B-ALL. In the AML phase, we again observed DNMT3A and TET2 mutations at high VAFs (>40%), as well as a PTPN11 p.A72T mutation (VAF 15%) and tetrasomy 8 that were undetectable at prior timepoints. A new TP53 p.R249T mutation was detected at the time of clinical progression after salvage chemotherapy, despite clearance of the PTPN11 mutated subclone. Cytogenetic analysis at diagnosis, first relapse, and second relapse confirmed MLL rearrangement t(4;11)(q21;q23). Conclusion: These results provide molecular evidence that primordial founder clones may persist during clinical remissions in MLL-rearranged leukemia and enable lineage switching upon relapse. More broadly, this case suggests that somatic mutations found in CHIP and other potentially pre-neoplastic states could confer increased risk of clonal escape from lineage-restricted therapies. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Genes encoding the alpha (GNAS) and beta (GNB1) subunits of the heterotrimeric G-protein complex are recurrently mutated in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Alterations in G-protein coupled receptors can affect signaling via the PI3K/AKT/mTOR and RAS/MAPK pathways, suggesting that GNB1/GNAS mutations may function similarly to mutations in RAS and tyrosine kinases in myeloid disease progression. However, unlike RAS mutations, GNB1/GNAS mutations are among the most commonly affected genes in clonal hematopoiesis of indeterminate potential (CHIP), suggestive of a distinct functional role in myeloid disease initiation. We evaluated 6343 unique patients who received gene panel sequencing at our institution as part of a diagnostic evaluation of known or suspected hematologic malignancy. We identified 68 patients that had at least 1 sample with a mutation in GNB1 (N =42), GNAS (N=24) or both (N=2) (Figure 1). Twenty-six patients had multiple samples (range 2-10) obtained at serial timepoints during the course of progression and treatment. GNB1 mutations affected codon 57 (K57E/M/N/T) in 38 out of 42 cases; the remaining were G53E, D76G, I81V, and K89R. GNAS mutations affected codon 201 (R201H/C) in 23 out of 24 cases. Forty patients had active myeloid neoplasms [AML (N=10), MDS (N=13), MPN (N=11), MDS/MPN (N=5), BPDCN (N=1)]. The distribution of myeloid diagnoses was similar among patients with GNB1 or GNAS mutations. Analysis of serial samples showed that GNB1/GNAS mutations were frequently acquired at the time of leukemic transformation and clinical progression. Among 5 patients with secondary AML, 2 patients acquired GNB1/GNAS mutations at the time of transformation from MDS to AML, and 3 patients were found to have GNB1/GNAS mutations in high-risk MDS prior to subsequent transformation to AML. After induction chemotherapy for AML, two patients had expanded or newly acquired GNB1 mutations at time of treatment failure. Notably, 15 out of the 40 (37.5%) patients with active myeloid disease had co-mutations in the MPN-associated genes JAK2, CALR, and MPL. Eleven of these patients had a clinical diagnosis of MPN, including myelofibrosis (n=5), essential thrombocytosis (n=3), polycythemia vera (n=2), and systemic mastocytosis (n=1). In the context of underlying oncogenic kinase alterations including JAK2 V617F, GNB1 mutations have been shown to promote resistance to kinase inhibitors in vitro. Fourteen out of fifteen patients had no history of ruxolitinib therapy, suggesting that these mutations arise during the course of disease progression in the absence of therapeutic selection pressures. The impact of GNB1/GNAS mutations on the clinical response to JAK inhibitors has not been evaluated. Twenty-eight patients in the cohort (41%) had no evidence of active myeloid disease, including 14 patients with a lymphoid malignancy [mature B-cell neoplasms (n=8), mature T cell neoplasms (n=3), plasma cell neoplasms (n=2), acute lymphoblastic leukemia (n=1)], 6 patients with AML in complete remission, and 8 patients without a clinical diagnosis of hematologic malignancy. Four of the AML patients had a GNAS or GNB1 mutation that was newly acquired or persistent at the time of complete remission following induction chemotherapy. All 4 patients underwent allogeneic stem cell transplant (HSCT) based on disease risk at diagnosis and are alive with median follow up of 2.5 years. In 1 patient, a new GNB1 mutation was identified immediately after HSCT in the context of 100% donor chimerism, suggesting donor-engrafted CHIP. All 8 patients without hematologic malignancy were evaluated for cytopenias or cytoses at the time of the identification of the GNB1/GNAS mutation. GNB1 and GNAS are recurrently mutated in diverse clinical and genetic contexts. In patients with active myeloid disease, MPN-associated mutations were common and GNB1/GNAS mutations were newly acquired at the time of clinical progression, suggesting a functional role similar to other gene mutations that activate mitogenic signaling pathways. However, many GNB1 and GNAS mutations are seen as sole mutations in the absence of active myeloid disease, suggesting that unlike other signaling mutations they can drive initiation of clonal hematopoiesis. The diverse clinical spectrum of patients with GNB1/GNAS mutations suggests that these mutations have distinct, context dependent effects in hematopoietic cells. Disclosures Kim: Aushon Biosciences: Consultancy; LabCorp, Inc.: Consultancy; Papgene, Inc: Consultancy. Lane:N-of-one: Consultancy; Stemline Therapeutics: Research Funding.

The ability of cells to undergo apoptosis in response to cellular stresses functions as a safeguard against tumorigenesis. Consequently, compromised apoptotic signaling must precede tumor development. To design effective therapies for cancer treatment, the underlying mechanisms that govern cell survival need to be understood. The anti-apoptotic proteins of the Bcl-2 family are key regulators of cell survival and are frequently overexpressed in malignancies, leading to increased cancer cell survival. Bcl-w, the closest anti-apoptotic relative to Bcl-2 and Bcl-xL, is essential for spermatogenesis, but was considered dispensable for other cell types. However, we recently uncovered a significant role for Bcl-w in B-cell survival and lymphomagenesis. Specifically, loss of Bcl-w conferred sensitivity to growth factor deprivation-induced B-cell apoptosis. In addition, apoptosis triggered by hyperproliferative signals from the Myc oncogene was accentuated by loss of Bcl-w. Consequences of elevated apoptosis were observed in vivo, as Bcl-w loss profoundly delayed Myc-mediated B-cell lymphoma development. We also determined Myc regulates Bcl-w expression through its transcriptional regulation of the miR-15 family. Importantly, evaluation of Burkitt lymphoma (BL) patient samples revealed BCL-W overexpression was highly selected for, and that targeting BCL-W using multiple methods induced apoptosis of BL cells. In addition, overexpression of BCL-W in BL cell lines conferred resistance to the BH3-mimetics ABT-737 and ABT-263 that inhibit anti-apoptotic BCL-2 family members. Furthermore, BCL-W was overexpressed in diffuse large B-cell lymphoma (DLBCL) and correlated with decreased patient survival. Analysis of multiple data sets of other aggressive and indolent B-cell lymphomas showed that BCL-W was frequently overexpressed in them as well. Collectively, our data indicate that BCL-W is an understudied contributor to B-cell lymphoma development and survival that appears to be as important as BCL-2 to B-cell lymphomas. In addition, BCL-W is a predictive biomarker that could improve prognostication and the development of more effective treatment strategies for B-cell lymphomas. Citation Format: Clare M. Adams, Ramkrishna Mitra, Jerald Z. Gong, Annette S. Kim, John K. Choi, Christine M. Eischen. BCL-W significantly contributes to B-cell lymphoma survival and development [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 02.

Imatinib has dramatically altered the natural history of chronic myelogenous leukemia (CML), with the majority of patients now experiencing long-term remission and improved survival. However, in addition to the well-described phenomenon of resistance to imatinib, typically due to point mutations, uncommon consequences (eg, the development of Philadelphia chromosome–negative clones) may infrequently occur. We report 2 cases of sudden BCR/ABL1 + blast crisis in patients with CML who had achieved complete hematologic remission with imatinib therapy but were obligated to discontinue therapy owing to pancytopenia. These sudden blast crises were unusual at 3 levels: first, they were of precursor T lymphoblastic lineage; second, they had a primary extranodal presentation without overt bone marrow involvement; and third, they developed after recent cessation of imatinib. These observations suggest that the occurrence of 2 such rare cases in a single institution within a 1-year time frame may reflect another unusual consequence of imatinib therapy.

Background Although advances in sequencing technologies have popularized the use of microRNA (miRNA) sequencing (miRNA-seq) for the quantification of miRNA expression, questions remain concerning the optimal methodologies for analysis and utilization of the data. The construction of a miRNA sequencing library selects RNA by length rather than type. However, as we have previously described, miRNAs represent only a subset of the species obtained by size selection. Consequently, the libraries obtained for miRNA sequencing also contain a variety of additional species of small RNAs. This study looks at the prevalence of these other species obtained from bone marrow aspirate specimens and explores the predictive value of these small RNAs in the determination of response to therapy in myelodysplastic syndromes (MDS). Methods Paired pre and post treatment bone marrow aspirate specimens were obtained from patients with MDS who were treated with either azacytidine or decitabine (24 pre-treatment specimens, 23 post-treatment specimens) with 22 additional non-MDS control specimens. Total RNA was extracted from these specimens and submitted for next generation sequencing after an additional size exclusion step to enrich for small RNAs. The species of small RNAs were enumerated, single nucleotide variants (SNVs) identified, and finally the differential expression of tRNA-derived species (tDRs) in the specimens correlated with diseasestatus and response to therapy. Results Using miRNA sequencing data generated from bone marrow aspirate samples of patients with known MDS (N = 47) and controls (N = 23), we demonstrated that transfer RNA (tRNA) fragments (specifically tRNA halves, tRHs) are one of the most common species of small RNA isolated from size selection. Using tRNA expression values extracted from miRNA sequencing data, we identified six tRNA fragments that are differentially expressed between MDS and normal samples. Using the elastic net method, we identified four tRNAs-derived small RNAs (tDRs) that together can explain 67 % of the variation in treatment response for MDS patients. Similar analysis of specifically mitochondrial tDRs (mt-tDRs) identified 13 mt-tDRs which distinguished disease status in the samples and a single mt-tDR which predited response. Finally, 14 SNVs within the tDRs were found in at least 20 % of the MDS samples and were not observed in any of the control specimens. Discussion This study highlights the prevalence of tDRs in RNA-seq studies focused on small RNAs. The potential etiologies of these species, both technical and biologic, are discussed as well as important challenges in the interpretation of tDR data. Conclusions Our analysis results suggest that tRNA fragments can be accurately detected through miRNA sequencing data and that the expression of these species may be useful in the diagnosis of MDS and the prediction of response to therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1929-y) contains supplementary material, which is available to authorized users.

Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested that murine neonatal host defense against infection could be compromised by immunosuppressive CD71+ erythroid splenocytes. We examined the impact of CD71+ erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71+ erythroid (CD235a+) cells in human neonates. Adoptive transfer or an Ab-mediated reduction in neonatal CD71+ erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immunosuppression of stimulated adult CD11b+ cells was not limited to neonatal splenocytes; it also occurred with adult and neonatal bone marrow. Animals treated with anti-CD71 Ab showed reduced splenic bacterial load following bacterial challenge compared with isotype-treated mice. However, adoptive transfer of enriched CD71+ erythroid splenocytes to CD71+-reduced animals did not reduce bacterial clearance. Human CD71+CD235a+ cells were common among cord blood mononuclear cells and were shown to be reticulocytes. In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71+ erythroid splenocytes under these experimental conditions suggests that the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 Ab treatment, rather than a reduction in immunosuppressive CD71+ erythroid splenocytes, was likely responsible for the reported enhanced bacterial clearance. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests that they may have a limited role in reducing inflammation secondary to microbial colonization.

The type 1 insulin-like growth factor receptor (IGF-1R) is often overexpressed on tumor cells and is believed to play an important role in anchorage-independent proliferation. Additionally, cell culture studies have indicated that IGF-1R confers increased resistance to apoptosis caused by radiation or chemotherapeutic agents. Thus, inhibitors of the intracellular kinase domain of this receptor may have utility for the clinical treatment of cancer. As part of an effort to develop clinically useful inhibitors of IGF-1R kinase, a novel class of pyrrole-5-carboxaldehyde compounds was investigated. The compounds exhibited selectivity against the closely related insulin receptor kinase intrinsically and in cell-based assays. The inhibitors formed a reversible, covalent adduct at the kinase active site, and treatment of such adducts with sodium borohydride irreversibly inactivated the enzyme. Analysis of a tryptic digest of a covalently modified IGF-1R kinase fragment revealed that the active site Lys1003 had been reductively alkylated with the aldehyde inhibitor. Reductive alkylation of the insulin receptor kinase with one of these inhibitors led to a similarly inactivated enzyme which was examined by X-ray crystallography. The crystal structure confirmed the modification of the active site lysine side chain and revealed details of the key interactions between the inhibitor and enzyme.

The 2008 WHO classification scheme of hematolymphoid neoplasms recognizes a category of myeloid and lymphoid neoplasms (MLNs) with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1.1 The postulated cell of origin for PDGFRA or FGFR1-rearranged diseases is a pluripotent progenitor capable of giving rise to myeloid neoplasms (myeloproliferative neoplasms and acute leukemias) and to lymphoblastic leukemia/lymphoma. Historically, PDGFRB translocations have not been associated with malignancies of the lymphoid lineage. Myeloid neoplasms with abnormalities of PDGFRB have been described to have hematologic features of chronic myelomonocytic leukemia (CMML), sometimes with eosinophilia, or as various other myeloproliferative and/or myelodysplastic neoplasms.2 In recognition of this distinction, the classification scheme designates specific categories of “MLNs” with PDGFRA and FGFR1 rearrangement, but omits the word “lymphoid” from the PDGFRB-associated entity.1 To call attention to the rare occurrence of lymphoid and mixed MLNs with abnormalities of PDGFRB, we present the clinical and pathological details of 2 cases.

Epstein Barr virus (EBV)-related hemophagocytic lymphohistiocytosis (EBV-HLH) is a form of acquired, infection-related HLH which typically represents a fulminant presentation of an acute EBV infection of CD8+ T cells with 30-50% mortality rate. Systemic EBV-positive lymphoproliferative disease of childhood (SE-LPD) is a rare T cell lymphoproliferative disorder predominantly arising in the setting of acute EBV infection, often presenting with HLH. Since both entities have been associated with clonal T cell populations, the discrimination between these diseases is often ambiguous. We report a unique case of a 21 years old female who presented with clinical and laboratory findings of florid HLH in the setting of markedly elevated EBV titers (>1 million) and an aberrant T cell population shown to be clonal by flow cytometry, karyotype, and molecular studies. This case raises the differential of EBV-HLH versus SE-LPD. Review of the literature identified 74 cases of reported EBV-HLH and 21 cases of SE-LPD with associated HLH in 25 studies. Of those cases with available outcome data, 62 of 92 cases (67%) were fatal. Of 60 cases in which molecular clonality was demonstrated, 37 (62%) were fatal, while all 14 cases (100%) demonstrating karyotypic abnormalities were fatal. Given the karyotypic findings in this sentinel case, a diagnosis of SE-LPD was rendered. The overlapping clinical and pathologic findings suggest that EBV-HLH and SE-LPD are a biologic continuum, rather than discrete entities. The most clinically useful marker of mortality was an abnormal karyotype rather than other standards of clonality assessment.

The Wiskott-Aldrich syndrome protein (WASP; encoded by the gene WAS) and its homologs are important regulators of the actin cytoskeleton, mediating communication between Rho-family GTPases and the actin nucleation/crosslinking factor, the Arp2/3 complex. Many WAS mutations impair cytoskeletal control in hematopoietic tissues, resulting in functional and developmental defects that define the X-linked Wiskott-Aldrich syndrome (WAS) and the related X-linked thrombocytopenia (XLT). These diseases seem to result from reduced WASP signaling, often through decreased transcription or translation of the gene. Here we describe a new disease, X-linked severe congenital neutropenia (XLN), caused by a novel L270P mutation in the region of WAS encoding the conserved GTPase binding domain (GBD). In vitro, the mutant protein is constitutively activated through disruption of an autoinhibitory domain in the wild-type protein, indicating that loss of WASP autoinhibition is a key event in XLN. Our findings highlight the importance of precise regulation of WASP in hematopoietic development and function, as impairment versus enhancement of its activity give rise to distinct spectra of cellular defects and clinical phenotypes.

The Rho-family GTPase, Cdc42, can regulate the actin cytoskeleton through activation of Wiskott-Aldrich syndrome protein (WASP) family members. Activation relieves an autoinhibitory contact between the GTPase-binding domain and the carboxy-terminal region of WASP proteins. Here we report the autoinhibited structure of the GTPase-binding domain of WASP, which can be induced by the C-terminal region or by organic co-solvents. In the autoinhibited complex, intramolecular interactions with the GTPase-binding domain occlude residues of the C terminus that regulate the Arp2/3 actin-nucleating complex. Binding of Cdc42 to the GTPase-binding domain causes a dramatic conformational change, resulting in disruption of the hydrophobic core and release of the C terminus, enabling its interaction with the actin regulatory machinery. These data show that 'intrinsically unstructured' peptides such as the GTPase-binding domain of WASP can be induced into distinct structural and functional states depending on context.

The asymmetric syntheses of 6-deoxyerythronolide B (1) and oleandolide (2) have been achieved, each in 18 linear steps. These syntheses demonstrate the utility of chiral β-keto imide building block 3 as a versatile building block for the aldol-based assemblage of polypropionate-derived natural products.

The synthesis of 6-deoxyerythronolide B ( 1 ) has been completed using the β-ketoimide dipropionyl building block 2 in the synthesis of the C 1 C 7 and C 8 C 15 subunits. A convergent double stereodifferentiating Mukaiyama aldol coupling reaction, a biomimetic deoxygenation, and a high-yield macrocyclization were employed in an 18-step synthesis of this natural product.

Fluorescence in situ hybridization for abnormalities common to myelodysplastic syndrome (MDS FISH) is often used with traditional karyotype in the diagnosis and monitoring of myeloid neoplasms. However, its value in these roles has been questioned. To evaluate its utility, we compared MDS FISH results with karyotype in 544 bone marrow specimens obtained for diagnosis (180 cases) or follow-up (364 cases) of myeloid neoplasia. We found excellent concordance between FISH and karyotype, such that FISH is rarely abnormal (1.7% at diagnosis and 3.0% at follow-up) in cases with normal karyotype. Even in the rare discordant cases, the abnormal FISH has little or no clinical value. Thus, we propose that this test should be limited to cases with inadequate karyotype only. Such guidelines could result in significant cost savings with no impact on patient diagnosis.

Myelodysplastic syndromes (MDS) are a group of hematopoietic malignancies characterized by ineffective hematopoiesis. Recently, we identified MDS-associated microRNAs (miRNAs) that are down-regulated in MDS. This study examines possible explanations for that observed down-regulation of miRNA expression in MDS. Since genomic losses are insufficient to explain the down-regulation of all our MDS-associated miRNAs, we explored other avenues. We demonstrate that these miRNAs are predominantly intragenic, and that, in many cases, they and their host genes are expressed in a similar pattern during myeloid maturation, suggesting their co-regulation. This co-regulation is further supported by the down-regulation of several of the host genes in MDS and increased methylation of the shared promoters of several miRNAs and their respective host genes. These studies identify a role of hypermethylation of miRNA promoters in the down-regulation of MDS-associated miRNAs, unifying research on miRNAs in MDS and epigenetic regulation in MDS into a common pathway.

Merkel cell carcinoma is an aggressive neoplasm of the skin that shows frequent lymph node metastases, but has only rarely been reported in the bone marrow. Herein we report a case of a 64-year-old male with a history of plasma cell myeloma and recent skin diagnosis of Merkel cell carcinoma who presented for a routine follow-up bone marrow to assess his myeloma. The biopsy showed persistent plasma cell myeloma, trilineage dysplasia, and clusters of neuroendocrine cells consistent with metastatic Merkel cell carcinoma. Discussion of this case, a review of metastatic Merkel cell carcinoma, and identification of clinical settings in which staging bone marrow biopsy may be warranted are presented.

Risk stratification of acute myeloid leukemia (AML) is important for prognosis and for guiding treatment decisions. The National Comprehensive Cancer Network (NCCN) classifies AML into three risk categories (favorable, intermediate, or poor) based on recurrent cytogenetic or molecular abnormalities. Clinical next-generation sequencing (NGS) is beginning to clarify the relationship between these categories and the spectra of oncogenic mutations, and leading to the development of new targeted therapies. The role of NGS data in favorable risk disease and the interaction of the NCCN classification with other prognostic risk factors such as age are not fully understood. Here, we assess 32 favorable risk [12 inv(16), 2 t(8;21), and 18 normal karyotype NPM1 mutated/FLT3 ITD negative] and 88 poor risk (33 transformed from documented MDS, 47 with complex or monosomal karyotype without previous documented MDS, and 8 therapy-related with normal karyotype) cases of AML. Results of NGS using a 37 gene, myeloid neoplasm-specific panel were incorporated with conventional cytogenetics and FISH results, as well as clinical history including patient age and survival data. Genes were arranged into four functional groups: splicing, epigenetic, signaling, and transcription (see Table 1). Analysis of the mutational spectra categorized by NCCN risk category and by age reveals several patterns (see Table 1). TP53 mutations were associated with a low total mutation number, as previously reported. Core binding factor (CBF) AMLs were primarily seen in young patients (

Objective The myelodysplastic syndromes (MDS) are aging-associated disorders characterized by ineffective maturation of hematopoietic elements, which are often diagnostically challenging. This study identifies microRNAs (miRNA) and miRNA targets that might represent diagnostic markers for MDS. Materials and Methods This study utilized a total of 42 MDS samples and 45 controls. A discovery set of 20 frozen bone marrow mononuclear cell samples (10 MDS, 10 controls) was profiled on a custom Agilent miRNA microarray. Classifier miRNAs were validated in a separate set of 49 paraffin-embedded particle preparations by real-time polymerase chain reaction (24 MDS, 25 controls). Target prediction analysis was compared to a de novo transcriptional profile of MDS derived from the Microarray Innovations in Leukemia study. c-Myb and Sufu were further investigated by immunohistochemical stains on a set of 26 paraffin-embedded samples. Results We identified 13 miRNAs of interest from the discovery set, 8 of which proved statistically significant on real-time polymerase chain reaction verification. These eight miRNAs were then examined in an independent real-time polymerase chain reaction validation set. Notably, hsa-miR-378, hsa-miR-632, and hsa-miR-636 demonstrated particularly high discrimination between MDS and normal controls. Target prediction identified potential targets of miRNA regulation that correspond to many of the genes that characterize MDS. Immunohistochemical staining performed on a third validation set confirmed that c-Myb and Sufu are differentially expressed in MDS. Conclusions Our data utilize both discovery and validation sets and two complementary platforms to identify miRNAs associated with MDS. We have analyzed predicted targets and identified c-Myb and Sufu as potential diagnostic markers of MDS.

The World Health Organization (WHO) Classification of Tumours of Haematopoietic and Lymphoid Tissue, 2008 edition, states that anaplastic large cell lymphoma (ALCL) is “consistently negative for Epstein-Barr virus (EBV)". The statement made by the WHO has led to the widespread belief that EBV can have no pathogenic role in ALCL. Herein we report a case of an immunocompetent 35-year-old male who presented with hemophagocytic syndrome secondary to lymphoma for which diagnostic material consisted solely of a bone marrow biopsy. The biopsy demonstrated large anaplastic cells which were uniformly positive for surface CD3, CD30 (strong membranous and Golgi expression), CD45, TIA-1 and Granzyme B but negative for ALK-1. In-situ hybridization was strongly positive for EBER in the large neoplastic cells. The uniformity of CD30 expression and positivity for cytotoxic markers on the anaplastic tumor cells raised the diagnostic possibility of an EBV-associated ALCL, ALK-. Discussion of this case as well as a retrospective review of 64 cases of reported of EBV+ ALCL are presented.

The stereochemical and heterofunctional complexity of the polypropionate-derived macrolide antibiotics poses a formidable challenge for stereoselective synthesis, and these target structures have provided the stimulus for the development of a host of new enantioand diastereoselective bond constructions.1 In this paper we illustrate, in the context of an efficient synthesis of oleandolide aglycon (1),2 how polypropionate chains may be rapidly assembled using the chiral â-ketoimide building block 2 and its associated aldol reaction methodology recently developed in these laboratories.3,4 As illustrated in Scheme 1, the synthesis plan relied upon â-ketoimide 2 for the construction of both the C1-C8 and C9C14 oleandolide fragments. Concurrent application of a sequential aldol reduction strategy to both fragments established 8 of the 10 requisite stereocenters, while an imide enolate alkylation reaction was employed to control the lone C6 stereocenter in the C5-C8 subunit. In the final stereoselective transformation, the introduction of the C8-epoxide with the desired stereochemistry was effected through the directed VO(acac)2/t-BuO2H epoxidation of the 9-(S)-allylic alcohol prior to macrocyclization.5,6 This last step becomes much more challenging to implement when it is postponed until after macrocycle construction as the two previous syntheses of oleandolide have revealed.2 The synthesis of the C1-C8 fragment began with the titaniummediated syn aldol reaction between aldehyde 47 and â-ketoimide 2 (Scheme 2).3a,8 This double stereodifferentiating reaction (eq 1) proceeded in excellent yield with high anti Felkin diastereoselection. Treatment of aldol adduct 5 with Zn(BH4)2 established the C5-hydroxyl stereocenter Via a chelate-controlled

The synthesis of 6-deoxyerythronolide B ( 1 ) has been completed using the β-ketoimide dipropionyl building block 2 in the synthesis of the C 1 C 7 and C 8 C 15 subunits. A convergent double stereodifferentiating Mukaiyama aldol coupling reaction, a biomimetic deoxygenation, and a high-yield macrocyclization were employed in an 18-step synthesis of this natural product.

The asymmetric syntheses of 6-deoxyerythronolide B (1) and oleandolide (2) have been achieved, each in 18 linear steps. These syntheses demonstrate the utility of chiral β-keto imide building block 3 as a versatile building block for the aldol-based assemblage of polypropionate-derived natural products.

(1; R1 = H, R2 = Bn) (S) [90719-32-7] C10H11NO2 (MW 177.20) InChI = 1S/C10H11NO2/c12-10-11-9(7-13-10)6-8-4-2-1-3-5-8/h1-5,9H,6-7H2,(H,11,12)/t9-/m0/s1 InChIKey = OJOFMLDBXPDXLQ-VIFPVBQESA-N (Li salt) [123731-35-1](2; R1 = H, R2 = Bn) (R)[102029-44-7] C10H11NO2 (MW 177.20) InChI = 1S/C10H11NO2.Li/c12-10-11-9(7-13-10)6-8-4-2-1-3-5-8;/h1-5,9H,6-7H2,(H,11,12);/q;+1/p-1/t9-;/m1./s1 InChIKey = UNWHDOBWJMTBIY-SBSPUUFOSA-M (Li salt) [128677-61-2](3; R1 = H, R2 = i-Pr) (S) [17016-83-0] C6H11NO2 (MW 129.16) InChI = 1S/C6H11NO2.Li/c1-4(2)5-3-9-6(8)7-5;/h4-5H,3H2,1-2H3,(H,7,8);/q;+1/p-1/t5-;/m1./s1 InChIKey = UMVXXAHAUGRFCT-NUBCRITNSA-M InChI = 1S/C6H11NO2/c1-4(2)5-3-9-6(8)7-5/h4-5H,3H2,1-2H3,(H,7,8)/t5-/m1/s1 InChIKey = YBUPWRYTXGAWJX-RXMQYKEDSA-N (Li salt) [96021-69-1](4; R1 = Ph, R2 = Me) (4R,5S) [77943-39-6] C10H11NO2 (MW 177.20) InChI = 1S/C10H11NO2.Li/c1-7-9(13-10(12)11-7)8-5-3-2-4-6-8;/h2-7,9H,1H3,(H,11,12);/q;+1/p-1/t7-,9-;/m1./s1 InChIKey = OOIADRZYWZFYQM-PRCZDLBKSA-M InChI = 1S/C10H11NO2/c1-7-9(13-10(12)11-7)8-5-3-2-4-6-8/h2-7,9H,1H3,(H,11,12)/t7-,9-/m1/s1 InChIKey = PPIBJOQGAJBQDF-VXNVDRBHSA-N (Li salt) [92061-65-7](5; R1 = Ph, R2 = Me) (4S,5R) [16251-45-9] C10H11NO2 (MW 177.20) InChI = 1S/C10H11NO2.Li/c1-7-9(13-10(12)11-7)8-5-3-2-4-6-8;/h2-7,9H,1H3,(H,11,12);/q;+1/p-1/t7-,9-;/m0./s1 InChIKey = OOIADRZYWZFYQM-KUSKTZOESA-M InChI = 1S/C10H11NO2/c1-7-9(13-10(12)11-7)8-5-3-2-4-6-8/h2-7,9H,1H3,(H,11,12)/t7-,9-/m0/s1 InChIKey = PPIBJOQGAJBQDF-CBAPKCEASA-N (Li salt) [127882-97-7](6; R1 = H, R2 = Ph) (S) [99395-88-7] C9H9NO2 (MW 163.18) InChI = 1S/C9H9NO2.Li/c11-9-10-8(6-12-9)7-4-2-1-3-5-7;/h1-5,8H,6H2,(H,10,11);/q;+1/p-1/t8-;/m1./s1 InChIKey = HRFWPCNUKHEZCG-DDWIOCJRSA-M InChI = 1S/C9H9NO2/c11-9-10-8(6-12-9)7-4-2-1-3-5-7/h1-5,8H,6H2,(H,10,11)/t8-/m1/s1 InChIKey = QDMNNMIOWVJVLY-MRVPVSSYSA-N (7; R1 = H, R2 = Ph) (R)[90319-52-1] C9H9NO2 (MW 163.18) InChI = 1S/C9H9NO2/c11-9-10-8(6-12-9)7-4-2-1-3-5-7/h1-5,8H,6H2,(H,10,11)/t8-/m0/s1 InChIKey = QDMNNMIOWVJVLY-QMMMGPOBSA-N (8; R1 = H, R2 = tert-Bu) (S) [54705-42-9] C7H13NO2 (MW 143.19) InChI = 1S/C7H13NO2/c1-7(2,3)5-4-10-6(9)8-5/h5H,4H2,1-3H3,(H,8,9)/t5-/m1/s1 InChIKey = WKUHGFGTMLOSKM-RXMQYKEDSA-N (chiral auxiliaries used in asymmetric alkylations,1 acylations,2 halogenations,3 aminations,4 hydroxylations,5 aldol reactions,6 conjugate additions,7, 8 Diels–Alder reactions,9 acyl transfer,10 and sulfinyl transfer11) Physical Data: 1 mp 87–89 °C; 2 mp 85–87 °C; 3 mp 71–72 °C; 4 mp 118–121 °C; 5 mp 118–121 °C; 6 mp 130–132 °C; 7 mp 130–132 °C; 8 mp 118–120 °C. Solubility: sol most polar organic solvents. Form Supplied in: white crystalline solid; commercially available. Analysis of Reagent Purity: 99% purity attainable by GLC. Handling, Storage, and Precautions: no special handling or storage precautions are necessary. There is no known toxicity. It may be harmful by inhalation, ingestion, or skin absorption and may cause skin or eye irritation.

Background Myelodysplastic syndrome (MDS) is a common disease of the elderly characterized by ineffective maturation of hematopoietic cells manifesting as low blood counts, morphologic atypia, and predisposition to the development of acute leukemia. The only FDA-approved drugs for the treatment of all subytpes of MDS are the DNA methyltransferase inhibitors (DNMTis), azacytidine and decitabine. However, only a minority of patients achieve a hematologic response or better on these agents. We have previously identified a miRNA signature which distinguishes MDS patients from normal controls. We sought to investigate whether miRNA expression might also be predictive of how patients respond to therapy with DNA methyltransferase inhibitors (DNMTis). Design We collected paired fresh frozen bone marrow mononuclear cells (BM-MNCs) from 28 patients with MDS both pre and post treatment with DNMTis as well as from 30 normal controls. Following enrichment for miRNAs and other small RNA species using the TruSeq Small RNA sample preparation kit (Illumina, San Diego, CA), miRNAs were sequenced on an Illumina HiSeq 2500. Alignment was performed using Bowtie 2 against reference sequences from mirBASE and differential expression analysis was analyzed using a rank sum score from the following analysis programs: DESeq, edgeR and baySeq. The response to the DNMTis was characterized based upon the following scale: 1 = complete remission, 2 = marrow complete remission, 3 = partial remission, 4 = hematologic improvement, 5 = stable disease, and 6 = progressive disease. Linear regression analysis was conducted using the response score as outcome and miRNA expression value as the predictor. Results Several differences were found between the control samples and the pre-treatment MDS samples, many of which have been described previously, including hsa-miR-125b, hsa-miR-342, hsa-miR-140, and hsa-miR-150. Not unexpectedly, the expression of numerous miRNAs was significantly altered as a result of treatment with DNMTis. However, there were no significant miRNA expression differences between responders and non-responders in the post-treatment samples and no significant differences between the changes in miRNA expression (pre versus post treatment changes) in responders and non-responders. Interestingly, 5 miRNAs demonstrated significant association between their pre-treatment expression and the degree of response (FDR < 0.05): hsa-miR-125a, hsa-miR-342, hsa-miR-146b, hsa-miR-146a, hsa-miR-423, and hsa-miR-150. These miRNAs share striking overlap in their mRNA targets including key pathway regulators in the p53 pathway as well as numerous growth factors implicated in hematopoietic stem cells as well as hematopoietic stem cell markers. Conclusion These studies confirm that miRNA expression differences differentiate between MDS and normal controls. The administration of DNMTis results in widespread expression changes regardless of the response to therapy. However, the expression of a small handful of miRNA are positively correlated with the degree of response in MDS pre-treatment samples to DNMTis and therefore may be useful as markers which predict responders to DNMTi therapy in addition to suggesting biological pathways implicated in the mechanism of response. These results await further prospective studies for confirmation. Disclosures No relevant conflicts of interest to declare.

Genomics has enabled the examination of the totality of disease at the transcriptome level. Dependent upon a myriad of genetic aberrations for its pathogenesis, cancer has been the focus of gene expression profiling studies that have highlighted the potential clinical applications of this technology. This type of molecular profiling has the potential to enhance the ability of pathologists and oncologists to correctly classify tumors, not just into existing subgroups which may or may not have clear prognostic implications, but into new groups which carry predictable correlations with outcomes. Ultimately, these outcome predictions can be tied to specific treatment regimens, allowing clinicians to predict at the time of diagnosis to which therapy a given patient may best respond. Although this ultimate goal of personalized therapy remains in the future, the numerous studies to date have clearly demonstrated the overall feasibility of this approach. This review will showcase a few of these studies in several key tumor types with the goal of demonstrating which type of studies have been conducted and what types of results are currently possible.

Current drug discovery efforts focus primarily on proteins with defined enzymatic or small molecule binding sites. Autoregulatory domains represent attractive alternative targets for small molecule inhibitors because they also occur in noncatalytic proteins and because allosteric inhibitors may avoid specificity problems inherent in active site–directed inhibitors. We report here the identification of wiskostatin, a chemical inhibitor of the neural Wiskott-Aldrich syndrome protein (N-WASP). Wiskostatin interacts with a cleft in the regulatory GTPase-binding domain (GBD) of WASP in the solution structure of the complex. Wiskostatin induces folding of the isolated, unstructured GBD into its autoinhibited conformation, suggesting that wiskostatin functions by stabilizing N-WASP in its autoinhibited state. The use of small molecules to bias conformational equilibria represents a potentially general strategy for chemical inhibition of autoinhibited proteins, even in cases where such sites have not been naturally evolved in a target.

New chiral imide enolate alkylation, aldol, and Michael addition bond constructions have been employed in the asymmetric synthesis of the C 26 -C 37 portion of calyculin A

8605 Background: MRD assayed by multi-parameter flow cytometer (MFC), has prognostic significance after HDT/ASCT for MM (Paiva et. al. 2008). The frequency of MRD negativity (-) after induction therapy using novel agents such as immunomodulatory drugs like lenalidomide (IMiDs), and proteasome inhibitors like bortezomib, is unknown. The impact of HDT/ASCT on MRD status in this patient group has not been studied. Methods: We performed a retrospective study of all MM patients undergoing HDT/ASCT (January 2010 - December 2012) in our institution. No restrictions on inclusion were made based on the International Myeloma Working Group response criteria. All patients had novel agents as part of their initial induction regimen. Statistical analysis was by SPSS software (V 12.0). MRD status was determined by MFC on bone marrow samples pre- HDC/ASCT [M1] and post- HDC/ASCT (D100 [M2] and I year [M3]). MFC was done with antibodies against CD45, CD19, CD138, CD38, CD20, CD56, and anti-k and l cytoplasmic antibodies. Results: MRD status was available on 91 patients pre-transplant. Of these patients, 80 had MFC recorded at M2 and 17 patients had MFC recorded at M3. Fifty-eight percent were male and 76% were Caucasian. Forty percent received IMiDs, while 60% got proteasome based therapies. Of the 91 patients with MRD pre-HDC/ASCT, 58% (53/91) were MRD (-), and of these patients 89% (41/46) remained MRD (-) at M2. 48 patients were MRD positive (+) pre-HDC/ASCT, 58% (20/34) became MRD (-) at M2. Age, cytogenetic risk, disease stage, number of chemotherapy cycles or immunofixation status had no impact on MRD status. There were only 6 relapses in the cohort, thus the impact of MRD status on progression-free survival could not be studied. Conclusions: Novel agents improve depth of response pre-transplant. HDC/ASCT increases MRD negativity post-transplant. MRD status could aid better timing of HDC/ASCT or adoption of a risk-adapted strategy for high-risk patients. MRD status validation in a prospective cohort is underway at our center (NCT01215344). With future follow-up, the impact of MRD on progression-free survival in the era of novel agents will be determined.

4208 Introduction: Metaphase cytogenetic (MG) studies are routinely performed on bone marrow aspirate specimens in patients with hematologic disorders. While more sensitive tests are available which target specific aberrations, MG remains an inexpensive method to identify both anticipated and unanticipated global changes. However, due to the occasional finding of non-clonal changes in single metaphases, the International System for Human Cytogenetic Nomenclature recommends that the abnormality be present in at least two cells (three cells for losses of whole chromosomes) before it is considered a clonal change. Noting a high frequency of single metaphase chromosomal abnormalities (SMCAs) in the heavily pre-treated population at Vanderbilt University Medical Center (VUMC), we sought to quantify the incidence of those SMCAs with minimal or no apparent clinical significance and to determine the best interpretation and reporting mechanism for such changes. Methods: Retrospective data was collected and analyzed from a 16 month period encompassing March 2010 to June 2011. During this time period, all cases of SMCAs were identified. For each instance all tandem ancillary studies (fluorescent in situ hybridization (FISH), molecular studies) as well as all prior and subsequent cytogenetic studies associated with bone marrow biopsies were analyzed. Results: A total of 2916 bone marrow biopsies were performed at VUMC during this 16 month time frame. Of these 2916 cases, 680 had abnormal karyotypes, including 151 instances of chromosomal aberrations which were identified in only a single cell out of at least twenty metaphases examined (SMCAs). These 151 instances corresponded to 133 patients. Compared to the overall distribution of bone marrow diagnoses/indications at VUMC, there were a disproportionately high number of cases of acute myeloid leukemia (AML)and/or myelodysplasia (MDS) (45% of SMCAs versus 31% of total VUMC bone marrow biopsies) and lymphoma (15% of SMCAs versus 5%) (chi-square test, p-value < 0.001). Across diagnoses, 79% of all cases were found in patients after chemotherapy or radiation treatment, with a very high association with prior therapy in cases of AML (94%), B lymphoblastic leukemia (100%), myeloproliferative neoplasms (100%), lymphoma (85%), and MDS (84%). Only cases for which the clinical indication was to rule out bone marrow failure had a significant percentage of SMCAs in untreated marrows, potentially representing stress hematopoiesis. Of the 151 cases of SMCA, 81 (60%) had a prior marrow, a subsequent marrow, or both which did not show the SMCA, suggesting a transient incidental finding of probable minimal clinical significance. An additional 29/151 (19%) cases were seen only once at VUMC, but the aberration had no known clear disease association and are likely of minimal clinical significance. The SMCA was likely indicative of minimal residual disease in only 13 cases (9%), confirmed by concordance with prior marrow findings or tandem FISH studies with known disease association. Only 4 of the 151 cases (2.6% of cases) were of unclear clinical significance at the time of the result but were later confirmed to be significant by identification of a similar finding in a subsequent marrow. Of the SMCAs believed to be transitory and therapy-related, the aberrations clustered with variable changes seen in chromosomes 1 (particularly t(1;19) and t(1;7)), 6, 7, 9, and 13 as well as frequent trisomies of chromosomes 18, 21, and 22. Conclusions: Retrospective analysis identified that 79% of SMCAs identified were of minimal or no clinical significance. Those cases in which the SMCA likely represents a true marker of disease represented only 9% of cases and were readily identifiable at the time of the marrow by correlation with prior cytogenetic findings or tandem FISH. In only 2.6% of cases was an SMCA only subsequently proven to be clinically significant. There was a high association with prior chemotherapy in these SMCA cases, and the cases have preferred “hotspots” of the therapy-related transient chromosomal changes, including numerous translocations. We therefore recommend the de-emphasis of SMCAs in post-therapy specimens by reporting a normal karyotype in the formal interpretation with only mention of the SMCA in a comment to enable correlation with future studies. This reporting scheme will help distinguish true clonal evolution from a transient laboratory finding. Disclosures: No relevant conflicts of interest to declare.

Abstract 1710 Introduction: Since the identification of mutations in RPS19 in patients with Diamond-Blackfan anemia (DBA) in 1999,1 proteins in ribosomal biogenesis have been implicated in other congenital bone marrow failure syndromes, including Schwachman-Diamond syndrome (SDS) (SBDS) and X-linked dyskeratosis congenital (DKC1). 2 Collectively known as ribosomopathies, these syndromes share an increased risk of the development of a myelodysplastic syndrome (MDS) and acute myeloid leukemia. MDS is an acquired bone marrow failure syndrome. In the best understood subtype of MDS, 5q- syndrome, deletions of RPS14 in the common deleted region of 5q appear to contribute to the anemia seen in this disease.3 We hypothesize that non-5q minus MDS may also be an acquired ribosomopathy syndrome. Methods: Using published gene expression profiling studies of SDS,4 DBA,5 and MDS,6 the dysregulated ribosomal proteins were compared and 5 ribosomal proteins were identified which represented a union between the three sets of data: RPS10, RPS14, RPS24, RPL14, and RPL36. For practical reasons, we chose to examine RPS14, RPS24, and RPL36. In addition, we selected DKC1, SBDS, CASP3, and Ki67 to further probe the involvement of potential pathways of ribosomal biogenesis, apoptosis, and proliferation. A total of 16 MDS paraffin-embedded bone marrow samples were examined as well as 10 control samples. Immunohistochemical analysis was performed on 4 μm sections from the bone marrow preparations. Staining was performed on the EnVision+ horseradish peroxidase system (DAKO, Carpinteria, CA), using anti-RPS14 (#16683-1-ap, Proteintech, Chicago, IL), anti-RPS24 (#hpa003364, Sigma-Aldrich, St. Louis, MO), RPL36 (#ab74737, Abcam, Cambridge, MA), anti-DKC1 (#nbp1-40097, Novus Biologicals, Littleton, CO), anti-SBDS (#LS-C40532, LifeSpan Biosciences, Seattle,WA), anti-caspase 3 (#mbs440008, MyBioSource, San Diego, CA), and anti-Ki67 (#M7240, Dako, Carpinteria, CA). Expression levels of the proteins were counted in 200 nucleated cells in a blinded fashion by two independent reviewers. A stain index for each sample was determined based upon percentage of positive staining cells per hematologic lineage using upon the counterstain morphology. Unpaired T-test analysis was used to ascertain significance of differences between MDS and normal control samples. Results: Statistically significant changes were found in the ribosomal proteins in at least one cell lineage. Surprisingly, SBDS was found to be expressed in an increased percentage of erythroid cells in MDS (positive cytoplasmic staining in 16% of erythroid cells versus 6% in controls, p value 0.0066). RPL36 was also found to have increased expression in the myeloid lineage in MDS (positive nuclear and cytoplasmic staining in 52% of myeloid cells in MDS versus 33% in controls, p value 0.025) as did RPS24 (positive cytoplasmic staining in 33% of myeloid cells in MDS versus 11% in controls, p value 0.005). RPS14 demonstrated increased expression in myeloid cells (positive cytoplasmic staining in 38% of myeloid cells in MDS versus 18% in controls, p value 0.0042) as well as erythroid cells (positive cytoplasmic staining in 18% of myeloid cells in MDS versus 8% in controls, p value 0.0091), but in fewer megakaryocytes (26% versus 75%, p value 0.038). DKC1 showed increased expression in myeloid nuclei (91% versus 78%, p value 0.043). However, there were no statistically significant differences in expression of caspase 3 or Ki67. Of note, RPS24 is overexpressed while its predicted regulator miR-342 has been shown to be underexpressed in MDS. Similarly, underexpression of miR-10b and miR-150 may correlate with overexpression of DKC1 and correspondingly miRs-378, -140, and -103 with SBDS. Conclusions: Unlike the congenital bone marrow failure syndromes which are associated with loss of function or haploinsufficiency of ribosomal proteins, there appears to be increased expression of ribosomal proteins in the maturing cells of MDS. In contrast to previous studies on CD34+ MDS cells which found decreases in ribosomal protein expression,6 these increases in expression were found on morphologically more mature cells. Several of the ribosomal proteins are predicted to be regulated by miRNAs previously shown to be decreased in MDS.7 There is no overt correspondence with alterations in either apoptosis (measured by caspase 3 expression) or proliferation (Ki67 expression). Disclosures: No relevant conflicts of interest to declare.

Abstract 2073 Introduction: Molecular and cytogenetic testing is critical in diagnosis and management of hematologic malignancies. These tests are expensive, complex, and frequently inappropriately ordered. There are no comprehensive guidelines to assist in the disease- and patient-specific selection of these tests. Traditionally, most tests are ordered before morphologic evaluation of the specimen, which could help guide test selection. We hypothesize that this leads to significant over-ordering of tests unnecessary for diagnosis or monitoring, as well as under-ordering of necessary tests. As part of the hematopathology diagnostic management team (DMT) effort, we evaluated this hypothesis by establishing evidence-based rules for molecular and cytogenetic test ordering that are applied by the pathologist after review of clinical history and bone marrow aspirate smears. Methods: Using published evidence and best clinical practices, a team of clinical hematologists and hematopathologists developed rules, termed standard operating procedures (SOPs), for selecting cytogenetic and molecular tests on bone marrow biopsies from adult patients performed for myelodysplastic syndrome/acute myeloid leukemia (MDS/AML), plasma cell myeloma, lymphoma, myeloproliferative neoplasms, lymphoblastic leukemia, and bone marrow failure. The rules specify testing in five clinical scenarios: diagnosis or follow-up with morphologically overt disease, bone marrow staging (for lymphoma), routine follow-up without overt disease, evaluation prior to stem cell transplant (pre-SCT), and follow-up after stem cell transplant (post-SCT). To determine the effect that application of these SOPs would have had on test ordering and results, bone marrow biopsies from adults (>18 years) were evaluated retrospectively over a six-month time period. Each test was ruled concordant or discordant with the corresponding SOP. Omitted tests, those recommended by the SOPs, but not performed, were also identified. Statistical analysis of concordant and discordant tests was performed using the Chi-square test. Results: A total of 3,007 tests (769 karyotypes, 1,790 FISH, and 448 molecular tests) were performed on 804 adult bone marrows. Of these, 1,080 (36%) were discordant with the SOPs. Discordant tests were most frequent in myeloma (471), MDS/AML (231), and lymphoma (215). The most common clinical settings for discordant tests were routine follow-up (403) and post-SCT (417). To determine if these tests were necessary, we determined the positive rate of both concordant and discordant tests. The positive rate was significantly higher in the concordant (490/1,929; 25%) than the discordant tests (38/1,078; 4%) (P Conclusions: Retrospective analysis of cytogenetic and molecular test ordering demonstrates that more than one-third of cytogenetic or molecular tests ordered on bone marrow specimens at a large tertiary care institution were discordant with rules (SOPs) based on published literature and best clinical practices. Furthermore, the vast majority (>99%) of these discordant tests were negative for any abnormality, redundant, or potential false positives, suggesting that the exclusion of these tests would have little negative clinical impact. In addition, there were a large number of omitted tests, seen in 18% of all marrows. This analysis predicts that institution of rules-based morphology-influenced test ordering would decrease uninformative testing, while increasing appropriate informative tests, leading to more cost-effective, accurate, and complete diagnosis and monitoring of hematolymphoid malignancies. We have therefore instituted a rules-based, morphology-directed approach at our institution. Disclosures: No relevant conflicts of interest to declare.