ALBERT

Description: Chromosome translocations are among the most common genetic abnormalities in human leukemia. Each translocation may affect a different pair of genes. The abnormally expressed genes that result from the different translocations provide a rich source for identifying specific markers for clinical diagnosis of each translocation. Microarrays have identified genes differentially expressed in different translocations but the results between laboratories are not always compatible. We used SAGE to quantitate gene expression in bone marrow (BM) samples from 22 patients with four types of AML, namely de novo AML M2 with t(8;21), AML M3 or M3V with t(15;17), AML M4Eo with inv(16), AML M5 with t(9;11) or secondary t(9;11).We generated SAGE libraries from CD15+ leukemic myeloid progenitor cells, collecting over 106 SAGE tags, of which 209,486 were unique tags; 136,010 were known genes and ESTs, and 73,476 were novel transcripts. SAGE tags for further analysis were selected based on a 5-fold difference between patients’ samples and normal CD15+ BM; they were also statistically significantly different at the 5 % level. Using these strict criteria, we identified 1,571 unique tags, of which 1,405 were known genes and ESTs, and 166 were novel transcripts that were either specific for each translocation or were common for all four translocations. Changes in expression of these known genes which fall into different gene ontogeny functional categories varied by translocation. For example, those associated with macromolecular biosynthesis, transport and transcription were most altered in the t(8;21); those related to defense response and apoptosis were altered in the t(15;17); cell proliferation genes were most affected by the t(9;11). Cell surface receptor signaling, intracellular signaling and RNA processing were altered in treatment related but not in de novo t(9;11). From this analysis, we identified the functional molecular signature of each translocation. We designed a custom microarray to validate our SAGE data analysis. Our initial pilot microarray experiment with 96 genes that were specific for each translocation or common for all translocations used mononuclear cells from normal and patient BM and translocation cell lines, ME-1, THP-1, Mono Mac-6, Kasumi 1, NB-4; the array data from BM matched the SAGE data for 48-75 % of genes and the majority of cell lines, except ME-1, matched at least 70 % with the SAGE results for the appropriate translocation. We have now designed a full-scale microarray that contains over 400 probes including 250 known genes, 61 ESTs, 45 novel sequences and 48 genes reported by others. We will test at least 100 patients’ samples with the four translocations to validate which genes provide a robust, reproducible “fingerprint” for each translocation and for all translocations. We will correlate our microarray data with age, sex, race, response to treatment, survival and other mutations (FLT3, MLL ITD, etc) to identify any transcripts that might reliably define these categories. Our results will provide new insights into genes that collaborate with each translocation to lead to a fully leukemic phenotype.

Description: Siglecs (sialic acid-binding immunoglobulin superfamily lectins) are a family of cell surface receptors involved in regulating the immune response. The CD33-Related Siglecs (CD33rSiglecs, namely Siglec-3, -5 through -11 and -XII in humans) are a subgroup of these molecules found primarily on cells of the innate immune system. All are type-1 transmembrane proteins with an N-terminal sialic acid-recognizing V-set domain followed by a variable number of C-2 set domains, a transmembrane region and a cytosolic C-terminal domain that includes two tyrosine-based signaling motifs. Available data suggest an inhibitory signaling role in the innate immune response, mediated by recognition of host sialic acids as “self”. Nine of the 13 known primate Siglec genes along with 14 Siglec pseudogenes comprise the CD33-related Siglec gene cluster on human chromosome 19. Gene conversion is a mechanism for copying part of a genomic sequence into another, contributing to genetic diversity. Pseudogenes are known to play role in generating functional diversity of related genes (e.g., antibody diversity via gene conversion in chickens). We recently analyzed genomic sequences of the CD33-related Siglec gene cluster in three primates (human, chimpanzee and baboon) and found evidence for rapid evolution in this gene family (Angata et al., PNAS, in press). Additional evolutionary studies using distance-based phylogenetic trees shows evidence for three partial gene conversions between Siglec genes and adjacent Siglec pseudogenes. All three involve the coding regions for extracellular domains that mediate sialic acid recognition, and two involve a pseudogene converting a known Siglec gene. Functional analyses using recombinant proteins show marked differences in sialic acid-binding properties between the converted Siglec and its non-converted ortholog. These findings suggest that gene conversion with pseudogenes has contributed to the rapid functional evolution of the Siglecs, and provides a novel mechanism for changing sialic acid binding specificity. We hypothesize that this mechanism allows for rapid evolutionary adjustments in the recognition of endogenous sialic acids as “self”, a potential factor in controlling the innate immune response.

Description: We have recently demonstrated the presence of Kaposi's sarcoma–associated herpesvirus (KSHV) in cultured bone marrow (BM) stromal dendritic cells from all patients with myeloma studied. To show that these findings were not an artifact of tissue culture, we performed in situ hybridization (ISH) and polymerase chain reaction (PCR) to detect KSHV in BM core biopsies. Using ISH to open reading frame-72 (ORF 72), we localized KSHV to BM dendritic cells in 17 of 20 patients with myeloma, 2 patients with plasmacytosis associated with the acquired immunodeficiency syndrome, and 1 case of aplastic anemia. In contrast, BM from normal subjects (n = 4) and patients with lymphoma and leukemia (n = 21) did not contain KSHV. PCR amplification with KSHV primers demonstrated product in fresh BM biopsy samples from 6 of 7 myeloma patients, whereas three normal marrows contained no amplified product. These findings suggest that KSHV, possibly through alterations in the BM microenvironment and production of viral interleukin-6 (vIL-6), may stimulate and maintain abnormal plasma cell proliferation in myeloma and related disorders.

Description: Objective: Although Fresh Frozen Plasma (FFP) is indicated in critically ill patients, there are growing concerns about it's cost effectiveness and adverse effects in instances that do not conform to evidence-based indications. Despite a range of adverse reaction with the inappropriate usage of FFP, its usage has significantly increased over the last decade. We previously evaluated the overuse of FFP in our community teaching hospital in 2010-2011 and 2016-2017. The interventions to reduce the inappropriate FFP transfusion were implemented after the results from 2016-2017. The present multicenter study is a post-interventional study that describes practices regarding administration of FFP in three community teaching hospitals between 2017-2018. Methods: A retrospective chart review of patients who received FFP transfusion in three community teaching hospitals between 2017-2018 were included in our study. Criteria were established to evaluate the appropriateness of FFP. Descriptive statistics were utilized to compare the appropriateness of FFP transfusion among the participating hospitals. Results: Overall, 306 participants were evaluated where 25% of the participants (77 out of 306) received inappropriate FFP transfusions. The most common rationale for inappropriate FFP transfusions were bleeding with INR less than 1.5 followed by INR over 1.5 (without any evidence of active bleeding).Based on our study criteria, the proportion of inappropriate FFP transfused out of the total FFP transfused decreased from 49% in the study period 2016-2017 to 25% in the study period 2017-2018. 15% of the study population received inappropriate FFP transfusion for bleeding in 2017-2018 compared to 42% in 2016-2017. Conclusion: Our study showed a reduction in inappropriate FFP transfusions in 2017-2018 compared to 2016-2017. This can be attributed to the interventions implemented after obtaining study results from 2016-2017. The interventions included creating fliers educating about lack of effectiveness of FFP to reduce INR below 1.6 and arranging educational talks from transfusion committee about the evidence-based usage of FFP transfusion. Such interventions are essential to reduce healthcare expenditure and transfusion related adverse events. Disclosures No relevant conflicts of interest to declare.

Description: Objective: Studies demonstrated that inappropriate usage of fresh frozen plasma (FFP) is associated with adverse reaction and poor health outcome in the hospitalized patients. Reducing inappropriate FFP administration in the inpatient settings can minimize potential for adverse events, and foster controllable cost expenditure. Guideline regarding indication of FFP transfusion is scarce. We aimed to assess the appropriateness of FFP transfusion in the setting of community teaching hospital. Methods: A retrospective chart review of patients received FFP transfusion in two community teaching hospitals between 2016-2017 were included in our study. Frequency of appropriateness of FFP transfusion was reported. We also reported percent increase from previous years to compare the FFP usage from 2010-2011 to 2016-2017. Results: Of 138 patients received FFP transfusion in 2016-2017, 62% (86 patients) received inappropriate transfusion. 18% of patients received FFP to correct high INR (〉1.6) requiring emergency surgery. 53% of the patients received inappropriate transfusion for bleeding in 2016-2017 compared to 25% in 2010-2011. There was 10% rise of inappropriate or overuse of FFP transfusion in 2016-2017 than 2010-2011. Conclusion: Inappropriate FFP transfusion is significantly associated with adverse health outcome and increased healthcare cost. This study justifies the need for continuous audit for appropriate use of FFP. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Although opioids are the recommended treatment for acute pain management in sickle cell disease (SCD) patients, the opioid epidemic may have negatively affected the care of these patients in the emergency care settings. Literature suggested that ED physicians routinely overestimate opioid misuse in patients with SCD. Pennsylvania mandated Prescription drug monitoring programs (PDMP) in August 2016 which may affect ED opioid prescription among SCD patients. We evaluated the impact of that PDMP on opioid prescription in the management of acute pain for patients with SCD. Method: The data collection took place in the two community hospitals (Mercy Fitzgerald and Mercy Philadelphia Hospital) in Philadelphia. Participants were adults (aged ≥18 years) diagnosed with SCD who triaged for pain in the ED was included in the study. We grouped the encounter by year of PDMP implementation: prior to September 2016 (January 2016-August 2016), and after September 2016 (January 2017-August 2017) to collect patient records of a 6-month period and analyzed the data on number of visits, treatment types, opioid dispensing and total morphine milligram equivalents (MMEs) drug prescriptions during the ED visit. Paired t-test and chi square test were used to compare pre- and post-intervention results. Repeat encounters within 7 days period were excluded in the study. Result: The study included 180 qualifying ED visits (92 pre-intervention; 88 post-intervention). PDMP intervention was not significantly associated with reductions in the with opioid prescribing or the amount of prescribed MMEs. The mean age is 32.7 and 58% were male; and all were of African American race/ethnicity. However, prescriptions for non-opioid analgesics increased significantly during the same periods (p

Description: Complementary and genomic DNA for the murine transferrin receptor 2 (TfR2) were cloned and mapped to chromosome 5. Northern blot analysis showed that high levels of expression of murine TfR2 occurred in the liver, whereas expression of TfR1 in the liver was relatively low. During liver development, TfR2 was up-regulated and TfR1 was down-regulated. During erythrocytic differentiation of murine erythroleukemia (MEL) cells induced by dimethylsulfoxide, expression of TfR1 increased, whereas TfR2 decreased. In MEL cells, expression of TfR1 was induced by desferrioxamine, an iron chelator, and it was reduced by ferric nitrate. In contrast, levels of TfR2 were not affected by the cellular iron status. Reporter assay showed that GATA-1, an erythroid-specific transcription factor essential for erythrocytic differentiation at relatively early stages, enhanced TfR2 promoter activity. Interestingly, FOG-1, a cofactor of GATA-1 required for erythrocyte maturation, repressed the enhancement of the activity by GATA-1. Also, CCAAT-enhancer binding protein, which is abundant in liver, enhanced the promoter activity. Thus, tissue distribution of TfR2 was consistent with the reporter assays. Expression profiles of TfR2 were different from those of TfR1, suggesting unique functions for TfR2, which may be involved in iron metabolism, hepatocyte function, and erythrocytic differentiation.

Description: We have recently demonstrated the presence of Kaposi's sarcoma–associated herpesvirus (KSHV) in cultured bone marrow (BM) stromal dendritic cells from all patients with myeloma studied. To show that these findings were not an artifact of tissue culture, we performed in situ hybridization (ISH) and polymerase chain reaction (PCR) to detect KSHV in BM core biopsies. Using ISH to open reading frame-72 (ORF 72), we localized KSHV to BM dendritic cells in 17 of 20 patients with myeloma, 2 patients with plasmacytosis associated with the acquired immunodeficiency syndrome, and 1 case of aplastic anemia. In contrast, BM from normal subjects (n = 4) and patients with lymphoma and leukemia (n = 21) did not contain KSHV. PCR amplification with KSHV primers demonstrated product in fresh BM biopsy samples from 6 of 7 myeloma patients, whereas three normal marrows contained no amplified product. These findings suggest that KSHV, possibly through alterations in the BM microenvironment and production of viral interleukin-6 (vIL-6), may stimulate and maintain abnormal plasma cell proliferation in myeloma and related disorders.

Description: Abstract SCI-16 The Human Genome Project's completion of the human genome sequence in 2003 was a landmark scientific achievement of historic significance. It also signified a critical transition for the field of genomics, as the new foundation of genomic knowledge started to be used in powerful ways by researchers and clinicians to tackle increasingly complex problems in biomedicine. To exploit the opportunities provided by the human genome sequence and to ensure the productive growth of genomics as one of the most vital biomedical disciplines of the 21st century, the National Human Genome Research Institute (NHGRI) is pursuing a broad vision for genomics research beyond the Human Genome Project. This vision includes facilitating and supporting the highest-priority research areas that interconnect genomics to biology, to health, and to society.Current efforts in genomics research are focused on using genomic data, technologies, and insights to acquire a deeper understanding of biology and to uncover the genetic basis of human disease. Some of the most profound advances are being catalyzed by revolutionary new DNA sequencing technologies; these methods are already producing prodigious amounts of DNA sequence data, including from large numbers of individual patients. Such a capability, coupled with better associations between genetic diseases and specific regions of the human genome, are accelerating our understanding of the genetic basis for complex genetic disorders and for drug response. Together, these developments will usher in the era of genomic medicine. Disclosures: No relevant conflicts of interest to declare.

Description: PIK3CG, which encodes the catalytic subunit p110γ of phosphoinositide 3-OH-kinase-γ (PI3Kγ), has been assigned to chromosome band 7q22, a region that is frequently deleted in myeloid malignancies. PI3Kγ-mutant mice have hematologic defects and are predisposed to colon cancer. On the basis of these data, PIK3CG was evaluated as a candidate myeloid tumor suppressor gene (TSG). PIK3CG was mapped by fluorescence in situ hybridization adjacent and telomeric to a commonly deleted segment defined previously in myeloid leukemias with breakpoints within 7q22. PIK3CG contains 10 exons and spans approximately 37 kilobases of genomic DNA. Forty leukemias with monosomy 7 or a del(7q) were screened for PIK3CG mutations. Two patients had missense variations affecting residue 859 in the N-terminal catalytic domain of the protein. This allele was also detected in unaffected parents and in 1 of 60 control alleles; it probably represents a polymorphism. PIK3CG is unlikely to act as a recessive TSG in myeloid leukemias with monosomy 7.