ALBERT

Description: Key Points RH genotyping of red cells may improve matching of patients and donors and reduce Rh alloimmunization. RH genotype matching may improve use of an African American blood donor inventory.

Description: Background RH gene variation is frequent in individuals of African descent and contributes to Rh alloimmunization among patients with sickle cell disease (SCD). Despite antigen matching for C, E, and K and transfusion from African American (AA) donors, patients with SCD continue to form antibodies against Rh antigens (Chou et al 2013, Blood, 122, 1062). RH genotyping of patients with SCD and selected African American (AA) donor groups has the potential to improve transfusion therapy. We developed a virtual matching program with groups of AA blood donors and patients with SCD to select blood based on RH genotype and tested whether a RH genotype matching strategy is feasible. Moreover, we evaluated the feasibility of considering patient and AA donor RH genotypes for individuals with SCD immunized to D and e antigens since providing D- and/or e- RBCs often exposes the patient to other antigens they may lack and results in further alloimmunization events. Methods RH genotypes were determined in 657 patients with SCD and 625 AA blood donors by RH BeadChip (BioArray Solutions) and manual AS-PCR or exon sequence analysis for alleles ambiguous or not detected by Beadchip. Automated virtual donor matching for each patient with SCD was performed considering RHD and RHCE separately and together. Customized scripts in a Filemaker Pro database counted exact genetic matches as well as used more ÒinclusiveÓ criteria to select donors with only RH genes that would not encode Rh antigens absent in the patient. Results The distributions of RHD and RHCE alleles in donors and patients were similar. Of 24 RHD genes detected in both groups, 5 (RHD*D, *DAU0, *deleted D, *weak D 4.0, and *inactive RHDψ) accounted for 91% of donor alleles and 90% of patient alleles. Of 23 RHCE genes, the 7 most common accounted for 92% of donor alleles, and 91% of patient alleles. Match results for patients with 〉 25 donor matches or no matches according to locus and stringency of criteria are summarized in the table. Percent of 657 pts with 〉 25 or no matches by locus and stringency of criteria TableMatch by:RHDRHCERHD and RHCECriteria〉25None〉25None〉25NoneExact68.2%4.4%41.7%5.0%9.9%19.0%Inclusive*100%0%76.7%0.91%61.8%1.4% *Inclusive match includes donors with only RH genes that would not encode Rh antigens absent in the patient. Thirty D+ individuals had formed anti-D and were subsequently transfused D- RBCs, accounting for 70% of units transfused to these individuals (9305 D- units of 13304 total units transfused). Twenty-three individuals had at least one conventional RHD allele (77%). Virtual matching revealed an average of 8 donors (1.3%, range 0 - 44) with exact genetic matches and 43 donors (6.9%, range 0 - 44) using ÒinclusiveÓ criteria, compared to 128 donors (20.5%, range 113 - 210) available if matching by serology for D, C, and E. Twenty-one e+ individuals (7 E+e+ and 14 E-e+) had anti-e, and were transfused e-E+ RBCs if anti-e was demonstrating in their serum. Four of the 14 E-e+ subsequently formed anti-E. Virtual matching revealed an average of 7 donors (1.1%, range 0 - 22) with exact genetic matches and 34 donors (5.4%, range 5 - 107) using ÒinclusiveÓ criteria, compared to 133 donors (21.3%, range 113 - 166) available if matching by serology for D, C, and E. Conclusions Automated selection of AA donors based on RHD, RHCE, or both would be feasible for the majority of patients with SCD given an adequate genotyped donor pool. These studies are necessary to determine the number of minority donors required to support patients with SCD with genetically Rh matched donor units. Compared to donor matching by serologic Rh types, genetic matching for this patient cohort may prevent exposure to a foreign RhD or RhCE protein, but does limit the number of donors versus standard phenotype matching for C and E. For patients immunized to Rh antigens, providing RH genotype matched units may decrease the need for D- donor units and avoid further alloimmunization. Future prospective studies are needed to determine whether transfusion with RH genotype matched donor units can decrease alloimmunization and improve transfusion safety for patients with SCD. Disclosures No relevant conflicts of interest to declare.

Description: The hematopoietic progenitor cell (HPC) has the potential to differentiate into multiple cell types. Deciphering transcriptional regulatory networks necessary for HPCs to undergo lineage commitment is critical. Toward this goal, we established a serum-free in vitro differentiation system for human HPCs using CD34−positive cells isolated from peripheral blood of normal donors. Cells were cultured in the presence of Epo, SCF and IL-3 for two-weeks, and assessed for total hemoglobin and percent fetal hemoglobin. Adult-derived HPCs express large amounts of Hb A after approximately one week of culture with little expression of Hb F. Transcriptome analysis, using Affymetrix Human Genome U133 Plus 2.0 Arrays on RNA from days 1, 3, 5, 7, 9, 11 and 13 of culture shows more than 1500 differentially-expressed (DE)(〉 2-fold up or down) genes, a subset of which were validated independently by real-time RT-PCR. Up-regulated erythroid-specific genes included those for blood group antigens (Kell, Duffy, Rhesus), membrane proteins (ankryin 1, α- and β-spectrin, Band 4.1, Band 4.2, Band 4.9, glycophorin A), receptors (transferrin and erythropoietin receptors), heme biosynthesis enzymes (ALAS, ALAD, UROS, UROD, CPOX, PPOX, FECH), and transcription factors important in globin-gene expression (EKLF, BKLF, NFE2, GATA1) as well as α-, β- and γ-globin mRNAs. Down-regulated genes included those expressed in hematopoietic stem cells (GATA2, CD34) and non-erythroid hematopoietic lineages (MHC class I and II genes, CD33, CD53). The DE genes were clustered into a limited number of expression patterns, suggesting coordinate regulation of genes within each cluster. The DE gene set was examined in silico for co-regulation using transcriptional regulatory network analysis employing PAINT v3.2 software (Vadigepalli R et al., OMICS.2003; 7(3):235–52). GATA binding sites were represented on 149 of the DE gene promoters. Several transcriptional regulatory elements (TRE) were statistically significantly enriched in the DE gene set when compared to the array gene set. These include TRE for AP-1, CREB, GATA-1 and Oct-1. Co-regulation of genes in the different gene clusters will be analyzed by PAINT and findings validated by examination of TRE:transcription factor interactions. These studies should help define timing of transcription factor occupancy in relation to epigenetic changes occurring prior to transcriptome alteration during differentiation of human erythroid cells.

Description: Fibrinogen Philadelphia, a hypodysfibrinogenemia described in a family with a history of bleeding, is characterized by prolonged thrombin time, abnormal fibrin polymerization, and increased catabolism of the abnormal fibrinogen. Turbidity studies of polymerization of purified fibrinogen under different ionic conditions reveal a reduced lag period and lower final turbidity, indicating more rapid initial polymerization and impaired lateral aggregation. Consistent with this, scanning and transmission electron microscopy show fibers with substantially lower average fiber diameters. DNA sequence analysis of the fibrinogen genes A, B, and G revealed a T〉C transition in exon 9 resulting in a serine-to-proline substitution near the γ chain C-terminus (S378P). The S378P mutation is associated with fibrinogen Philadelphia in this kindred and was not found in 10 controls. This region of the γ chain is involved in fibrin polymerization, supporting this as the polymerization defect causing the mutation. Thus, this abnormal fibrinogen is characterized by 2 unique features: (1) abnormal polymerization probably due to a major defect in lateral aggregation and (2) hypercatabolism of the mutant protein. The location, nature, and unusual characteristics of this mutation may add to our understanding of fibrinogen protein interactions necessary for normal catabolism and fibrin formation.

Description: Renal insufficiency is the source of significant morbidity and mortality in adults with sickle cell disease (SCD). A population of adult patients with SCD and end-stage renal disease (ESRD) requiring hemodialysis was examined for factors that predict ESRD. ESRD patients (mean age 36.8 +/− 7.74) were compared with patients without ESRD over 50 years old (mean age 59.3 +/− 8.31). We found that the ESRD population was characterized by more males (14 males, 8 females ESRD vs. 4 males, 15 females non-ESRD), a higher occurrence of the CAR beta-like globin cluster haplotype (44 % ESRD vs. 11 % non-ESRD) and relatively lower fetal hemoglobin levels (3.6 +/− 2.16 ESRD vs. 8.6 +/− 6.35 non-ESRD). In order to further examine potential predictors of renal disease, including genetic modifiers unlinked to the beta-globin cluster, we examined a broader population of patients with elevated creatinine, defined as levels greater than 1.0 mg/dl, who did or did not require hemodialysis. We hypothesize that gender, beta-like globin cluster haplotype, fetal hemoglobin levels and polymorphisms in critical regulators of vasomotor tone are predictors of renal insufficiency in SCD. Genotyping of a single nucleotide polymorphism (SNP) in endothelial nitric oxide synthase (eNOS or NOS3) and an insertion/deletion polymorphism (I/D) in the angiotensin-converting enzyme (ACE) gene will allow us to determine if these polymorphisms are over-represented in this cohort. The eNOS T-786C SNP alters the level of transcription of the eNOS gene and the amount of eNOS activity. We recently showed that this polymorphism was associated with a history of acute chest syndrome in females with SCD (Br. J. Haematol., 2004, 124:240). The ACE I/D polymorphism has been associated with altered plasma ACE levels. Twenty-one adult patients with sickle cell disease with renal insufficiency and 25 age- and gender-matched control patients seen at Thomas Jefferson University are being examined. Genotype results and statistical analysis will be presented. It is hoped that identification of predictors of renal insufficiency in patients with SCD might lead to early identification of at-risk patients, allowing for therapeutic intervention.

Description: Abstract 1068 Background: Patients with AML who are eligible for chemotherapy are traditionally treated with infusional cytarabine and an anthracycline. CR rates with this combination have been approximately 50–60% with an induction mortality of 10–25%. However, this treatment is less effective in older patients in terms of CR attainment, remission duration, and overall survival. We present a retrospective analysis of an induction regimen that was designed based on the concept of timed sequential therapy. An initial pulse of chemotherapy is administered to eradicate cells in S phase. This is followed by a rest period during which previously quiescent cells that enter the cell cycle can be targeted by a second pulse of chemotherapy. The regimen incorporates high dose cytarabine, which has been shown to improve remission duration when used in induction, and dose-intensified anthracycline therapy, which has been shown to improve outcomes in younger patients. This report highlights the responses and tolerability of the study regimen, particularly in elderly patients and patients with prior myelodysplastic syndrome (MDS). Patients and Methods: One hundred sixty six patients were treated with timed sequential chemotherapy from 1998–2009. The treatment consisted of two doses of cytarabine 2 gm/m2 IVPB over 3 hours administered 12 hours apart followed by one dose of mitoxantrone 30 mg/m2 IVPB over 1 hour on days 1 and 5. Data on pre-therapy cytogenetics and MDS was collected for each patient. Remission status was assigned per the IWG response criteria for AML. Results: Median age of the patients was 54.5 years (range 17–85). There were eighty males and eighty-six females. Out of 166 patients, 11 (6.6%) patients had favorable, 83 (50%) had intermediate, and 72 (43.4%) had unfavorable karyotypes. One-third of the patients (57 pts) had AML transformed from MDS. The overall response rate (ORR: CR+CRi+CRp) for all patients was 69.9%. In patients who had de novo AML, the ORR was 79.8%, regardless of age. Patients over age 60 with de novo AML had an ORR of 74%. For those patients under the age of 60, the ORR was 82%. The ORR for patients with transformed AML was 52.6% (53% in pts over age 60, 52% in pts less than 60). The 30 day mortality rate was 3.4% (6/166). Five of the six patients who died had an unfavorable karyotype with 2 patients having therapy-related AML. The 30 day mortality for patients older than 60 was 3.3% (2/61) and for all patients was 3.6% (6/166). The 60 day mortality rate in all patients was 10.8% (18/166). Of the additional 12 patients, seven died from progressive disease and only 3 died of suspected therapy-related complications. Grade 3/4 hematologic toxicities were the most common adverse events seen. Conclusion: This is a convenient, 2-day induction regimen that is well-tolerated with comparatively low 30 and 60 day mortality and high response rates in older patients and those patients with AML transformed from MDS. This would be an excellent initial regimen for remission induction in a select population of patients in whom novel consolidation or maintenance therapies can be incorporated to further improve outcome. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1069 Background: Cytogenetic data plays an important role in assessing prognosis and determining choice of therapy for AML. Traditionally, patients with AML are treated with infusional cytarabine and an anthracycline. CR rates with this regimen have been reported at 50–60%. Evaluation of novel treatment regimens for AML should include determination of the impact of the regimen on intermediate and unfavorable cytogenetics. We present a retrospective analysis of a 2-day remission induction regimen, based on the concept of timed sequential therapy. The regimen combines high dose cytarabine, which has been shown to improve remission rates when used in induction therapy, and dose intensified anthracycline therapy, which has been shown to improve outcome in younger patients. The cycling cells are eradicated during an initial pulse of therapy, then, previously quiescent cells are targeted during the second pulse of therapy. Here we present the analysis of the study regimen and the response rates of patients with intermediate and unfavorable cytogenetic profiles. Patients and Methods: One hundred fifty five patients categorized as having intermediate or unfavorable cytogenetics were treated with timed sequential chemotherapy from 1998–2009. The treatment regimen consisted of two doses of cytarabine 2 gm/m2 IVPB given over 3 hours, administered 12 hours apart. This was followed by one dose of mitoxantrone 30 mg/m2 IVPB over 1 hour on days 1 and 5. Pre-therapy cytogenetic data was collected for each patient. Responses to therapy were determined based on IWG response criteria for AML. Results: One hundred fifty five patients with intermediate and unfavorable karyotypes received high dose cytarabine and mitoxantrone for remission induction therapy. Median age of patients was 55 years (ranged from 17–85). Sixty patients were 60 years or older. Eighty three patients (53.5%) had intermediate and 72 (46.5%) had unfavorable karyotypes. The younger patients (under 60 years of age) with intermediate cytogenetics achieved the following responses: 34 CR, 6 CRi, and 2 CRp. The overall response rate (ORR) was 80.8% for these younger patients, while the ORR for the older patients (over 60 years of age) with intermediate cytogenetics was 77.4% (15 CR, 4 CRi, 5 CRp). In the unfavorable cytogenetic category, the ORR of the younger patients (under 60 years of age) was 60.5% with 13CR, 8 CRi, and 5 CRp, while an ORR of 44.8% was shown in the older patients (over 60 years of age) with 9 CR, 1 CRi, and 3 CRp. Overall, twenty two out of seventy two (30.5%) had CR in the unfavorable group, 9 (12.5%) had CRi, and 8 (11%) had CRp for an overall response rate of 54%. The 30 day mortality rate was 3.8% (6/155). The 60 day mortality rate was 11.6%. The most common adverse events were Grade 3/4 hematologic toxicities. Conclusion: This convenient, 2-day induction regimen leads to high response rates with low treatment-related mortality in older patients and patients with unfavorable cytogenetic characteristics. Based on the tolerability and effectiveness, this regimen could potentially be useful in high risk transplant-eligible patients for remission induction. It appears that this regimen would also be appropriate for initial cytoreduction in elderly patients with AML prior to introduction of novel therapeutic strategies, such as hypomethylating agents or oral clofarabine for consolidation and maintenance. Disclosures: No relevant conflicts of interest to declare.