Peripheral WBC Research Articles

Aberrant Hedgehog Pathway Activity Marks Clinical MDS Progression and Accelerates Leukemic Transformation in Vivo

Introduction:Myelodysplastic Syndromes (MDS) represent a heterogeneous group of hematopoietic stem cell (HSC) disorders with varying clinical outcomes, but prognosis uniformly worsens with transformation to secondary acute myeloid leukemia (AML). Despite recent progress in genomics, the mechanisms responsible for disease progression are not fully understood as most of the somatic mutations defined thus far can be found at early stages of the disease. Previous studies have identified aberrant activation of the Hedgehog (Hh) signaling pathway in a subset of AML patients and the expression of the Hh-regulated transcription factor GLI2 correlated with inferior overall and progression free survival. In solid tumors, Hh pathway activation has been associated with metastatic disease progression, and we examined its role in MDS progression and transformation to AML.Methods/Results: We initially quantified changes in Hh pathway activity in CD34+ cells isolated from serial bone marrow samples collected from MDS patients at the time of diagnosis and following progression to AML. We found that the expression of the Hh target genes GLI1 and PTCH1 was increased in 67% of patients (4/6) suggesting that pathway activation was involved in the development of secondary AML. We also analyzed gene expression in 135 MDS patients and found significantly higher GLI2 expression in high-risk MDS (N=80) compared to low-risk MDS (N=55) (p=0.036). In addition, bone marrow blast percentage was significantly higher in the MDS cohort with higher GLI2 expression (mean±SEM=7.1±0.7%) than with lower expression (5.4±0.5%, p=0.039).In order to mechanistically study the effects of Hh pathway activation on MDS progression, we studied mice expressing the Nup98-HoxD13 (NHD13) fusion gene under the control of the vav promoter that generates progressive cytopenia and, in some animals, progression to AML. We crossed NHD13 mice with mice conditionally expressing the constitutively active mutant of the Hh signal transduction regulator Smoothened (SmoM2) in hematopoietic cells expressing Mx1-Cre. The survival of double transgenic animals (NHD13/SmoM2) was significantly shorter compared to mice expressing NHD13 (median survival of 3 months vs. 12 months, p<0.001, Fig. 1). Similar to previous reports we found that the expression of SmoM2 alone had no significant effect on survival or hematologic phenotype. Compared to NHD13 mice, NHD13/SmoM2 mice had significantly higher peripheral blood WBC (93.9k vs. 5.2k, p<0.001) and splenomegaly (633mg vs. 462mg, p=0.006) at the time of disease progression. Furthermore, an immature population of myeloid cells (Mac1+/Gr1 dim) was widely present in peripheral blood, bone marrow and spleen of NHD13/SmoM2 mice. We also examined mice prior to the onset of disease (4-8 weeks post Smo M2 induction) and found an increased frequency of myeloid cells (Mac1+/Gr1+) within the peripheral blood compared to wild type and NHD13/Smo M2 mice (wild type – 17.2%, NHD13 - 40.5%, NHD13/SmoM2 - 68%, p<0.01).Within the bone marrow, hematopoietic stem/progenitor cells (Lin-Sca1+cKit+) were depleted within both NHD13 and NHD13/SmoM2 mice. To examine the impact of Hh activation on clonogenic growth and self-renewal potential, we plated bone marrow cells in methylcellulose and found that NHD13/SmoM2 cells contained a significantly higher number of CFU compared to NHD13 cells (37 vs. 2, p=0.002) that was sustained with subsequent passages (400 vs. 30, p<0.001). In order to define the compartment enriched for leukemia initiating potential (LIC) we transplanted Lin- and Lin+ leukemic fractions from NHD13/SmoM2 mice into naive recipients and found that Lin+ cells led to death within 3 months suggesting that the Lin+ compartment has acquired self- renewal potential.Conclusions:We found that the Hh signaling pathway may be aberrantly activated in a subset of secondary AML patients progressing from MDS. We also demonstrated that activation of Hh signaling induces fatal AML in a mouse model of MDS characterized by inferior survival and widespread expansion of immature myeloid cells. This disease progression is driven by the acquisition of self-renewal and tumor initiating potential in differentiated Lin+ hematopoietic cells. Our findings suggest that Hh pathway inhibition may be a promising approach for AML arising from MDS. [Display omitted] DisclosuresMaciejewski:Alexion: Speakers Bureau; Celgene: Speakers Bureau.

The Prevalence of Extramedullary AML Detected By 18-FDG/PET-CT: Results from the Prospective PET-AML Trial

Introduction: Acute myeloid leukemia (AML) may present with either concomitant or isolated extramedullary (EM) AML. Data on the prevalence of EM AML are based on retrospective and clinical analyses which rely on findings from physical examination only or on coincidental findings in standard imaging procedures and range from 2.5 to 9.1% (Bakst et al., Blood 2011;118(14):3785-3793). Previous studies identified EM AML as a prognostic factor in patients with AML. 18Fluorodesoxy-Glucose Positron Emission Tomography – Computed Tomography (18FDG/PET-CT) is able to detect highly metabolic tissue and has proven efficacy in imaging studies for various types of malignant diseases. In a pilot study in patients with histologically proven EM AML, we were able to demonstrate a sensitivity of 90% using 18FDG/PET-CT imaging and found additional EM sites in 60% of the patients (Stölzel et al., Haematologica 2011;96(10):1552-1556). The aim of this prospective, observational study was to determine the prevalence of EM AML in patients with newly diagnosed or relapsed AML using 18FDG/PET-CT (ClinicalTrials.gov Identifier: NCT01278069).Patients and Methods: A total of 94 patients with AML (newly diagnosed AML, n = 85 and relapsed AML, n = 9) underwent total body 18FDG/PET-CT scans at diagnosis after giving informed consent. Patients with EM diagnosed by 18FDG/PET-CT underwent a second 18FDG/PET-CT scan after induction chemotherapy. The median age of all patients was 61 years (range, 27 to 79 years). Patients were included only if a delay of ≤ 5 days of initiation of induction- or re-induction chemotherapy was clinically justifiable to perform the study. 18FDG/PET-CT scans were performed using a Siemens Sensation 16 as part of a biograph (Siemens, Knoxville, TN, USA) with i.v. application of 18FDG (range of activity 223 to 433 MBq) and 120 ml i.v. contrast media Ultravist 370 (Bayer Schering Pharma, Leverkusen, Germany). Adverse reactions due to the application of i.v. 18FDG and i.v. contrast media did not occur.Results: Total body 18FDG/PET-CT imaging detected highly metabolic manifestations suggestive for EM AML in 21 patients (22%). During follow-up after induction chemotherapy, 18FDG-avid EM was still observed in 50% of all PET-positive patients. In comparison with PET-negative patients, those with PET-positive EM AML had a higher bone marrow blast count, a higher prevalence of FAB M5 morphology, higher peripheral WBC, higher serum LDH, and less frequent FLT3-ITD mutations. Sites of EM AML were connective tissue (n=4), parenchymal tissues (n=8), and lymph nodes (n=15). In patients with clinically overt EM AML (n=8) additional EM manifestations were detected in 62% (n=5). In 13 patients, samples from EM sites were obtained for histology review – in 10 patients histology confirmed the occurrence of EM AML in these sites, indicating a specificity of 77%. Importantly, in the remaining patients in whom histology could not confirm EM AML, concomitant malignant tumors were found. Extrapolating these results on the entire cohort, the prevalence of EM AML in newly diagnosed and relapsed AML is 17%.Conclusions: Our study is the first to prospectively evaluate 18FDG/PET-CT imaging for the diagnosis of EM AML. According to our results, 18FDG/PET-CT is a useful and safe tool in order to detect EM AML with a high specificity. We found a prevalence of EM AML of 17%. This is two- to eightfold higher, than in previously reported clinical studies. Integration of 18FDG/PET-CT-based imaging procedures as adjunct for response assessment in these patients seems to be important. Further development of other PET-based imaging procedures and integration in the setting of relapse after allogeneic hematopoietic stem cell transplantation might be necessary and will contribute to a better understanding of the biology and prognostic role of EM AML and the development of targeted treatment strategies in patients with AML. DisclosuresNo relevant conflicts of interest to declare.

Effects of lesion of posterior part of cerebellar vermis and fastigial nuclei on some immune responses in male rats

Considering the autonomic function of cerebellum, it is a potential neural area for immunomodulation. Though the role of fastigial nucleus (FN) and vestibulocerebellum on immunomodulation has been investigated, the results are contradictory. In the present study the role of posterior cerbellar vermis (PV) and FN on some immunological parameters such as TC and DC of WBC, leukocyte adhesive inhibition index (LAI) of splenic mononuclear cell (MNC) and phagocytic activity of peripheral WBC by fluorescence activated cell sorter (FACS) were investigated after two and three weeks lesions of PV (lobules VII, IX and X) and FN by aspiration technique in male rats. The TC of WBC was decreased after two weeks of PV+FN lesioned rats compared to that of sham lesion and control rats. The percentage neutrophil and eoisinophil counts were increased and the lymphocyte count was decreased after two weeks of PV+ FN lesioned rats compared to that of sham operated and control rats. The phagocytic activity of blood WBC and LAI of splenic MNC were increased after two weeks of PV+FN lesioned rats compared to that of sham lesioned and control rats. After three weeks of PV+FN lesioned these immunological parameters were regain to the normal level. The results indicate that the combined lesion of PV and FN induced enhanced immune responses but this effect is transitory.

Lack of clinical association and effect of peripheral WBC counts on immune cell function test in kidney transplant recipients with T-cell depleting induction and steroid-sparing maintenance therapy

The Cylex ImmuKnow assay measures the amount of stimulated ATP production by CD4+ T-cells, and has been used clinically, trying to predict rejection and infection episodes. However, predictive values of this assay after induction therapy with steroid-sparing maintenance protocols are unclear. In this single-center cohort study, we analyzed renal transplant recipients who received T-cell depleting+/−anti-IL2 receptor antibodies and tacrolimus/mycophenolate maintenance without steroids. A total of 4224 ImmuKnow levels in 306 patients were available for analysis. ImmuKnow levels (Mean±SE) changed over time after induction therapy with a paradoxical initial increase: 419±23, 461±32, 519±14, 411±10, 344±6, and 405±3 for pre-transplant, 0–1wk, 1wk–1mo, 1–3mos, 3mos–1yr, and thereafter. This change was parallel to the evolution of peripheral WBC counts and ImmuKnow levels had weak but significant correlation with WBC counts (R2=0.264, P<0.0001). The levels for biopsy-proven rejection (389±56) and borderline/clinical rejection (254±41) were not significantly higher than the levels of quiescent patients. The levels for opportunistic infection (349±48) and other infections (345±27) were not significantly lower than the levels of quiescent patients. The longitudinal changes in ImmuKnow levels were not predictive of rejection or infection. In conclusion, ImmuKnow levels can vary after T-cell depleting induction therapies at various time points, even without significant clinical events. Since ImmuKnow levels seem to be affected by WBC counts, ImmuKnow results need to be interpreted with caution. The effects of leukocytosis or leukopenia caused by immunosuppressive medication on the ImmuKnow assay need further investigation.

Optimal Time to Start Peripheral Blood Stem Cell Collection in Children with High-Risk Solid Tumors

In order to clarify the optimal timing for peripheral blood stem cell (PBSC) collection, PBSC collection records of 323 children who were scheduled to undergo autologous stem cell transplantation from two study periods differing in the timing of PBSC collection were analyzed. In the early study period (March 1998 to August 2007, n=198), PBSC collection was initiated when the peripheral WBC count exceeded 1,000/µL during recovery from chemotherapy. Findings in this study period indicated that initiation of PBSC collection at a higher WBC count might result in a greater CD34+ cell yield. Therefore, during the late study period (September 2007 to December 2012, n=125), PBSC collection was initiated when the WBC count exceeded 4,000/µL. Results in the late study period validated our conclusion from the early study period. Collection of a higher number of CD34+ cells was associated with a faster hematologic recovery after transplant in the late study period. Initiation of PBSC collection at WBC count > 4,000/µL was an independent factor for a greater CD34+ cell yield. In conclusion, PBSC collection at a higher WBC count is associated with a greater CD34+ cell yield, and consequently a faster hematologic recovery after transplant.

FLT3-ITD Internal Tandem Duplication Confers Poor Prognosis In Patients With Acute Promyelocytic Leukemia Treated With The AIDA Protocols. Long-Term Follow-Up Analysis

Although previous studies in APL have revealed that the FlT3-ITD mutation is associated with an elevated presenting WBC count, hypogranular variant (M3v) morphology and the short (bcr3) isoform of PML-RARA, the prognostic significance of FlT3 mutations in APL has not been firmly established. We report an update of these patients with a median follow-up of 9 years in which we observed that the presence at baseline of the FlT3-ITD mutation confers a very poor overall survival (OS). One hundred and forty-seven patients with newly diagnosed APL were observed and treated with the AIDA (73 patients) and AIDA2000 protocols (74 patients) at the Sapienza University of Rome during the period April 1993-October 2010. Diagnosis was initially established morphologically and subsequently confirmed in all cases by RT-PCR. The following clinical characteristics at diagnosis were analyzed according to the FlT3 status: age, sex, FAB classification, peripheral WBC and platelet count, hemoglobin, karyotype, PML/RAR isoform and relapse risk. For statistical analysis, the Wilcoxon-Mann-Whitney test was used to compare non-parametric series and the Fisher's exact test to compare categories. OS was estimated using the Kaplan-Meier method, whereas relapse-free considered as events relapse and death in CR. Thirty-three patients were identified as FlT3+, 19 were males (57%) and 14 were females; 27% had a variant type according to the FAB classification and 36% were classified as high-risk. Twenty-one FlT3+ patients (63%) presented the bcr3 transcript compared to 37/114 (32%) FlT3- patients (p=0.002). Eight FlT3+ patients (24%) experienced a differentiation syndrome compared to 14 (12%) in the negative cohort (p=0.02). After a median follow-up of 9 years (range 5-19), we could document a significant worse long-term outcome for FlT3+ patients: OS was 96% in the FlT3- cohort compared to 39% in the FlT3+ cohort (p=0.0001), relapse-free survival (RFS) 90% in FlT3- patients vs 30% in the FlT3+ ones (p=0.017), CIR at 9 years was 4% (95% CI, 1,279-6,238) for the FLT3-negative cohort and 60% (95% CI,22.632-81.236) for the FLT3-positive subset (p=0.0001). While this study confirms that there is no difference in response to induction with the AIDA schedule in FlT3+ APL patients, the longest follow-up so fare reported has allowed to demonstrate the significantly worse long-term outcome for this subset of APL, in terms of OS, RFS and DFS. Further studies aimed at investigating whether other clonal mutations may play a role in this unfavorable subset of patients are needed. Disclosures:No relevant conflicts of interest to declare.

Plerixafor, G-CSF and Azacitidine For The Treatment Of MDS: Results Of a Phase I Trial

Azacitidine has been shown to prolong survival and delay progression to leukemia in intermediate-2 and high risk myelodysplastic syndrome (MDS) in a randomized study compared to best supportive care. The combination of G-CSF and plerixafor is synergistic in increasing the release of stem and progenitor cells from the bone marrow through disruption of critical bone marrow stromal interactions including the CXCR4 / CXCL12 axis. The interaction of bone marrow stromal cells with the MDS tumor clone may play a roll in pathogenesis and response to treatment. We hypothesized that resistance of MDS to azacitidine may be related to MDS tumor and BM-stromal cell interactions, and disruption of these interactions by treatment with plerixafor + G-CSF could enhance sensitivity to azacitidine, thus improving complete and partial response rates.We conducted a phase I trial to investigate the safety and tolerability of plerixafor + G-CSF in combination with azacitidine in adult (18 years or older) MDS patients. Secondary objectives included response rates and biologic correlates evaluating: kinetics, phenotype, cell cycle status and kinetics of mobilization of MDS blasts compared to normal stem cells in select patients with informative cytogenetics. Major inclusion criteria included MDS defined by WHO criteria, 5 – 20% blasts on bone marrow aspirate, and at least one cytopenia in one cell lineage. Subjects receiving prior hypomethylating therapy were allowed. A standard 3+3 trial with 3 cohorts (320, 440, and 560 mcg/kg/day SC) was conducted. Dose limiting toxicity was defined as grade 3 or higher non-hematologic toxicity and hematologic toxicity of leukostasis or tumor lysis. Myelosuppression, infection, grade III nausea, fatigue, weight loss and electrolyte abnormalities were not considered dose limiting. Subjects initially received G-CSF 10 mcg/kg subcutaneous (SC) daily D1 – D8, plerixafor SC daily D3 – D8 and azacitidine 75mg/m2 SC D3 – D8, 4 hours after plerixafor administration. The trial was amended after the first 3 subjects to reduce G-CSF dose and administration to 5 days. ResultsTwo of the first three subjects enrolled in cohort 1 (320 mcg/kg/d plerixafor) had leukocytosis. The trial was amended to reduce the G-CSF dose (10 mcg/kg to 5 mcg/kg) and duration (8 days to 5 days) because of this. One subject had symptoms of leukostasis with a WBC reaching nearly 100K/uL and a subsequent subject developed hyperleukocytosis (WBC = 80K/uL) without leukostasis. The trial was amended to reduce the G-CSF to 5 days concurrent with plerixafor and azacitidine along with defining dose holding parameters for G-CSF and plerixafor if the peripheral blood WBC exceeded 40K/uL or if absolute blast count exceeded 10K/uL. Since amendment of the trial, 64 subjects have been screened and 20 subjects have been enrolled and are evaluable. Subjects included 65% males, median age 67, and MDS diagnosis at study entry including 6/18 (33%) RAEB-1 and 12/18 (67%) RAEB-2. 5/18 subjects (28%) had plerixafor and G-CSF held during treatment because of leukocytosis. 9/18 subjects (50%) had received no prior treatment for their MDS. DLTs were experienced in Cohort 1 related to thrombocytosis (n =1) and in Cohort 2 related to atrial fibrillation (n = 1) with near syncope. Major non-hematologic grade 3 or 4 adverse events included epistaxis, hypocalcemia, GI bleed, headache, dyspnea, infection with neutropenia, and bone pain. The MTD was determined to be 560mcg/kg plerixafor SC with no subjects (n = 6) in this cohort experiencing a DLT. The median number of cycles completed was 3. Reasons for stopping treatment included progression to leukemia (n = 6), physician choice (n = 2), withdrawal of consent (n = 1), adverse event (n = 2). Best response in those evaluable after completing 2 cycles of treatment (n = 14) showed marrow CR in 5/14 (36%, 3 in those not previously treated for MDS), stable disease in 5/14 (36%) and progressive disease 4/14 (29%). ConclusionPlerixafor plus G-CSF in combination with azacitidine was well tolerated in the studied MDS patients when given over 5 days and may be associated with encouraging response rates. Correlative studies are ongoing to evaluate changes in cell cycle, apoptosis and preferential mobilization of blasts using this regimen. We are currently enrolling an expanded cohort of 7 subjects at the MTD dose to evaluate preferential mobilization of blasts with plerixafor alone in cases using informative cytogenetics. Disclosures:Schroeder:Celgene: Research Funding; Sanofi Oncology: Research Funding. Off Label Use: Plerixafor and G-CSF for the treatment of MDS. Welch:Eisai: Research Funding. Stockerl-Goldstein:Millennium: Speakers Bureau; Celgene : Speakers Bureau.

Lupus mesenteric vasculitis: Clinical features and associated factors for the recurrence and prognosis of disease

ObjectiveTo evaluate the clinical characteristics of lupus mesenteric vasculitis (LMV) and identify the potential factors and appropriate treatments that are associated with disease relapse and prognosis in LMV. MethodsA retrospective cohort study was performed among patients admitted to the First Affiliated Hospital of Sun Yet-sen University between 2002 and 2011. Demographic information, clinical symptoms, laboratory findings, imaging characteristics like abdominal CT scan, ultrasonography, medications including corticosteroid, cyclophosphamide, and other immunosuppressive agents, and outcomes were documented. The endpoints of the study were defined as occurrence of severe complications that needed surgical intervention, disease recurrence, or death. ResultsOut of 3823 systemic lupus erythematosus (SLE) patients, 97 were diagnosed with mesenteric vasculitis with the overall prevalence of 2.5%. Among these 97 LMV patients, 13 died because of serious complications (13/97, 13.4%) and 2 presented intestinal perforation during the induction therapy stage. The logistic regression multivariate analysis indicated that leukopenia [peripheral WBC, odds ratio (OR) = 0.640, 95% confidence interval (CI): 0.456–0.896, P = 0.009], hypoalbuminemia (serum albumin, OR = 0.891, 95% CI: 0.798–0.994, P = 0.039) and elevated serum amylase (OR = 7.719, 95% CI: 1.795–33.185, P = 0.006) were positively associated with the occurrence of serious complications, while intravenous cyclophosphamide (CYC) therapy inhibited the occurrence of serious complications (OR = 0.220, 95% CI: 0.053–0.903, P = 0.036). A total of 79 patients who achieved remission were followed-up for 2–96 months and 18 cases experienced disease relapse (18/79, 22.8%). The statistical analysis adjusted by Cox proportional hazards models indicated that high-dose CYC therapy (≥1.0g/m2/month) was a protective factor for disease relapse and led to better outcomes [hazard ratio (HR) = 0.209, 95% CI: 0.049–0.887, P = 0.034], while the severe thickness of the bowel wall (>8mm) was a risk factor (HR = 7.308, 95% CI: 1.740–30.696, P = 0.007). LMV and lupus cystitis occurred concurrently in 22 (22/97, 22.7%) patients, and the symptoms of urinary tract resolved after treatment with corticosteroid and immunosupressants. ConclusionLMV is one of the serious complications of SLE with high mortality. The current study demonstrated that leukopenia, hypoalbuminemia, and elevated serum amylase were associated with severe adverse events, while CYC therapy led to better outcomes during remission-induction stage. Severe thickness of the bowel was a risk factor while high-dose CYC therapy was a protective factor for disease relapse in intensification therapy stage. It is necessary to evaluate the urinary tract involvement once LMV is diagnosed due to the frequent coexistence of these 2 diseases.

Successful Medical Management of Gastric Pneumatosis with Associated Portal Venous Air

Purpose: An 80-year-old woman with afib, DM, COPD, and cardiomyopathy was admitted with dyspnea and bilateral pneumonia. She was treated with meropenem IV, and a few days later, she began to complain of epigastric pain and nausea. The gastrointestinal symptoms progressed, and a CT scan revealed scant air in the main portal vein (Figure 1), gastric pneumatosis (Figure 2) and mild gastric distention. GI and surgical consultations were requested. Abdominal exam showed mild epigastric tenderness without signs of peritoneal irritation. Blood work revealed a normal peripheral WBC and a normal serum bicarbonate level. She was kept NPO, metronidazole and esomeprazole IV were added to her medical regimen and ngt suction was instituted. The patient remained clinically stable, her abdominal pain and nausea subsided and a repeat CT scan 48 hours later revealed resolution of the gastric pneumatosis and PV air. NGT suction was then discontinued and an oral diet was allowed. H. pylori stool antigen test was negative. The patient made a full clinical recovery and was discharged 2 weeks later. Outpatient EGD, performed 10 weeks later, revealed mucosal scarring in the gastric antrum.Figure: Axial CT demonstrating air in portal vein (circle).Figure: Axial CT demonstrating gastric pneumatosis (circle).Conclusion: Symptomatic gastric pneumatosis with PVG usually presages the need for surgical intervention. This case suggests that under the proper clinical scenario, with a thoughtful multidisciplinary approach, experienced clinicians may successfully manage this problem non-operatively, and there is not always a mandate for surgical intervention.

Molecular characterization of canine BCR‐ABL–positive chronic myelomonocytic leukemia before and after chemotherapy

Genetic aberrations linked to tumorigenesis have been identified in both canine and human hematopoietic malignancies. While the response of human patients to cancer treatments is often evaluated using cytogenetic techniques, this approach has not been used for dogs with comparable neoplasias. The aim of this study was to demonstrate the applicability of cytogenetic techniques to evaluate the cytogenetic response of canine leukemia to chemotherapy. Cytology and flow cytometric techniques were used to diagnose chronic myelomonocytic leukemia in a dog. High-resolution oligonucleotide array comparative genomic hybridization (oaCGH) and multicolor fluorescence in situ hybridization (FISH) were performed to identify and characterize DNA copy number aberrations (CNAs) and targeted structural chromosome aberrations in peripheral blood WBC at the time of diagnosis and following one week of chemotherapy. At the time of diagnosis, oaCGH indicated the presence of 22 distinct CNAs, of which trisomy of dog chromosome 7 (CFA 7) was the most evident. FISH analysis revealed that this CNA was present in 42% of leukemic cells; in addition, a breakpoint cluster region-Abelson murine leukemia viral oncogene homolog (BCR-ABL) translocation was evident in 17.3% of cells. After one week of treatment, the percentage of cells affected by trisomy of CFA7 and BCR-ABL translocation was reduced to 2% and 3.3%, respectively. Chromosome aberrations in canine leukemic cells may be monitored by molecular cytogenetic techniques to demonstrate cytogenetic remission following treatment. Further understanding of the genetic aberrations involved in canine leukemia may be crucial to improve treatment protocols.

Concurrent polycythemia vera and chronic lymphocytic leukemia: Treatment response to pegylated interferon alpha 2a

Our patient is a 55-year-old male initially diagnosed at age 45 (September 2002) with polycythemia vera (PV). At that time, the CBC showed a Hct of 72%, WBC 11.0/µl, and platelets of 560,000/µl. Phlebotomy treatments resulted in a decrease of the hematocrit to the normal range and a concomitant rise in the platelet count to above 1,000,000/µl. Additional laboratory studies at diagnosis demonstrated erythropoietin level <2.5 U/ml. Subsequent bone marrow biopsy demonstrated marrow cellularity of 70–80%, 1+ reticulin, stainable iron graded as decreased to absent, moderate left shift in myeloid maturation, negative Trichrome staining, and no atypical lymphoid infiltrate. For the subsequent nine years, no complications were reported and phlebotomy was required three to four times annually to maintain a hematocrit <45%. In 2008, the JAK2 V617F allele mutation burden was 65%. During this time period, the WBC count gradually rose to between 25 and 30,000/µl with no immature granulocytes noted. The platelet count plateaued at 1,500,000–1,600,000/µl. The spleen size increased to 16–17 cm. In 2010, an atypical lymphoid aggregate was noted on a bone marrow specimen. Subsequent flow cytometry of the peripheral blood suggested a monoclonal B-cell lymphocytosis. Shortly thereafter, the peripheral WBC count rose to 32,500/µl and the absolute lymphocyte count to 10,300/µl. Flow cytometry demonstrated 70% of the lymphocytes were B-cells positive for CD20 and CD5, and negative for CD10 and CD38. A diagnosis of B-cell chronic lymphocytic leukemia (CLL) RAI stage 0 was made. Additional studies demonstrated IgG VH segment percent homology at 92.70%. FISH analysis was normal. ZAP-70 analysis was indeterminate. In December 2010, the patient experienced a spontaneous hemorrhage in the left calf. After 30 days of hydroxyurea treatment, the platelet count decreased from 1,628,000 to 876,000/µl, and the WBC decreased from 32,500 to 22,500/µl. At this point, the hydroxyurea was discontinued and pegylated interferon alpha 2a (Pegasys) was begun at 45 µg subcutaneously weekly. Over 22 months, at an average Pegasys weekly dose of 72 µg, the WBC decreased from 30 to 7.0K, the absolute lymphocyte count from 10.4 to 2.3, and the platelets decreased to 450K. Phlebotomy occurred twice yearly. Peripheral blood findings demonstrated decrease of JAK-2 allelic ratio V617F/wild type to 30–40%, the monoclonal B-cell population to 12%, and Zap-70 status from indeterminant to negative. Abdominal ultrasound showed decrease in spleen size to 15 cm and bone marrow biopsy from 2012 demonstrated no appreciable change in reticulin (graded as mild). The bone marrow biopsy continued to show lymphoid involvement. Interferon-α is a nonleukemogenic treatment which has been shown in a number of recent studies to yield high rates of hematologic and molecular response in PV with limited toxicity 1. The ability of Pegasys to induce complete molecular responses suggests selective targeting of the malignant clone 2. Earlier clinical studies have likewise demonstrated a beneficial effect in early stage B-CLL using nonpegylated interferon alpha 2a 3. The diagnosis of concomitant CLL/MPN is a rare event and in a recent study analysis of prognostic markers for CLL demonstrated that in patients with a coexistent MPN, lymphoproliferative disorders follow a clinically indolent course 4, 5. Our patient has achieved a substantial hematologic, molecular and splenic response in his PV after 22 months on low dose Pegasys. Moreover, he has achieved a favorable reduction in the B-cell CLL clonal population and a change in Zap-70 status from indeterminate to negative. As far as we know, this is the first report of treatment of concomitant PV and chronic lymphocytic leukemia with Pegasys and the first to demonstrate a positive therapeutic effect on both disorders. H. Andrew Selinger1* Enrique Ballesteros2 Robert Bona3 1Prohealth Physicians, Bristol, Connecticut 06010 2University of Connecticut Health Center 3Frank Netter School of Medicine Quinnipia University

The synthesis of Tempol–phenol derivatives and their protection against radical-induced damage

Stable nitroxides are potential antioxidant drugs. In this study, we have linked nitroxide (Tempol) to phenol with the aim of improving radioprotective activity. The radical scavenging activities of four Tempol–phenol conjugates were evaluated in L-02 and MCF-7 cells survival assays. A differential protection in normal cells as compared to tumor cells was confirmed. In addition, the protective effect of these compounds was demonstrated in a greater increase in peripheral WBC, ANC, AMC, and platelet counts in an in vivo rat model. It might be possible to develop them into a possible agent for radical injury.

Chest CT features and outcome of necrotizing pneumonia caused by Mycoplasma pneumoniae in children (report of 30 cases)

To summarize the chest CT features and outcome of necrotizing pneumonia (NP) caused by Mycoplasma pneumoniae in children and to review the changes of common inflammatory parameters in NP patients to help clinicians understand the proper timing of CT scan. The imaging data from 30 cases of Mycoplasma pneumoniae pneumonia in NP group and 24 cases with non-necrotizing Mycoplasma penumoniae pneumonia (control group) were analyzed retrospectively. The changes of common inflammatory parameters in NP group and control group were compared. (1) The chest CT findings of NP (30 cases): 28 cases showed unilateral pneumonia, and 20 cases showed single lobar consolidation, 10 cases had multiple lobes involvement; pulmonary cavities were seen in 27 cases. There were decreased enhancement areas in the consolidation (22 cases). (2) The dynamic changes of CT signs during follow-up: The CT scan performed during the 1 - 2 months after onset of disease (23 cases) showed that pulmonary consolidation in 2 cases (9%) were absorbed, 18 cases (78%)had cavities in lung, 16 cases (70%) had pleural thickening, 2 cases (9%) atelectasis and 1 case (4%) bronchopleural fistula;the CT scan performed during the 2 - 3 months after onset of disease (11 cases) showed pulmonary consolidation in 7 cases (64%) were absorbed, 10 cases (91%) pleural thickness, 7 cases (64%) with cavities in lung, 5 cases (45%) atelectasis, 2 cases (18%) pulmonary lobe cysts and 1 case bronchopleural fistula. The CT scan performed at 3.5 years of disease course (10 cases) showed that there were no pulmonary consolidation in any of the cases, 4 cases had atelectasis, 4 cases had pulmonary cysts, and 1 case had band-like scars. (3) There were significant differences between NP group and control group in the maximum peripheral blood WBC, proportion of neutrophil and C-reactive protein(CRP, mg/L) (P < 0.01, 0.01, 0.001, respectively), and there was significant difference between the 2 groups in the duration of fever, abnormal WBC(d) and CRP(d) (P < 0.001). The chest CT features of NP caused by Mycoplasma pneumoniae in children were single lobular consolidation in most cases, NP had decreased parenchymal enhancement and cavity in the consolidation, and recovery was slow, the outcome included recovery, atelectasis or lobar cystic degeneration. The clinicians should pay more attention to the common inflammatory parameters when they suspect the Mycoplasma pneumoniae pneumonia is progressing into necrosis and make correct decision for chest CT examination.

Peripheral WBC Telomere Length as a Marker of Reproductive Age in Women

Reproductive aging is a continuous process that begins many years prior to menopause. The primary mechanism of reproductive aging is through the process of ovarian primordial follicle depletion, a process that exhibits considerable variation between women. The relationship between somatic and reproductive aging in women has been controversial, with some investigations suggesting a correlation between the two processes, whereas others have suggested no relationship. Peripheral lymphocyte telomere length is widely considered to be a marker of somatic aging.

Experimental Evidence That Granulocyte Transfusions Are Efficacious in Treatment of Neutropenic Hosts with Pulmonary Aspergillosis

Although polymorphonuclear leukocytes (PMNs) are powerfully anti-Aspergillus, transfusion therapy remains controversial, with conflicting results, and experimental support has been lacking. We devised a pulmonary infection model in neutropenic BALB/c mice, used an antibacterial regimen to prevent confounding sepsis, and optimized PMN induction, purifications, and dose. Mice were given 150 mg/kg cyclophosphamide every 4 days and a gentamicin-vancomycin-clindamycin-imipenem regimen daily beginning 4 days before intranasal challenge with 5 × 10(5) Aspergillus conidia. This regimen produced leukopenia (~10% of normal white blood cell [WBC] count; ≤ 10% PMNs) for 10 days, without bacterial superinfection. PMN donors given 100 μg/kg recombinant murine granulocyte colony-stimulating factor (G-CSF) for 10 days yielded 11 × 10(7) to 13.6 × 10(7) WBC/ml (81 to 87% PMNs). Infected mice were given PMN transfusions intravenously. In 2 experiments with up to 70% mortality of neutropenic controls, transfusion of 10(7) PMNs 1 and 4 days after challenge had negligible effects on peripheral WBC counts but improved survival (P = 0.007, 0.02), decreased lung CFU (P = 0.03, 0.005), and cleared infection in 28 to 50% of survivors. Transfusion of 5 × 10(6) PMNs showed partial protection. Transfusions given every other day did not improve protection. Our present results provide an experimental basis for enthusiasm for PMN transfusions in the therapy of aspergillosis in humans.

Differences in Acute Phase Reactants between Gout and Pseudogout

Objectives: To define clinical differences in the acute phase response and serum acute phase reactants between gout, pseudogout and crystal-induced arthritis in the presence of non-articular infections (CAI). Patients and Methods: Eleven patients with definite gout, 12 patients with pseudogout and 5 patients with CIA were included in the study. Results: The erythrocyte sedimentation rate (ESR) was significantly different between gout (68.2 ± 49.9 mm/Hr) and CIA (113.8 ± 37.2 mm/Hr) but not between gout and pseudogout (83.9 ± 45.6 mm/Hr) or between pseudogout and CIA. The C-reactive protein (CRP) was significantly increased between gout (10.1 ± 7.9 mg/dL) and pseudogout (18.9 ± 9.8 mg/dL), gout and CIA (36.5 ± 12.4 mg/dL) as well as between pseudogout and CIA. The peripheral white cell count was significantly different between gout (9.27 ± 3.7 k/μL) and CIA (16.5 ± 6.8 k/μL), and between pseudogout (8.9 ± 3.2 k/μL) and CIA. Conclusions: Measurement of ESR and CRP are helpful in crystal-induced arthritis. The CRP has more discriminating utility than the ESR in distinguishing between gout, pseudogout and CIA. Peripheral wbc is most useful for differentiating crystal-induced arthritis from CIA.

Dynamic Regulation of 5-Hydroxymethylcytosine At the γ-Globin Promoter During Erythroid Differentiation

AbstractAbstract 824DNA methylation is a key element responsible for γ-globin gene repression in adult erythroid cells. Our laboratory previously observed that the γ-globin gene promoter region was demethylated in a progressive manner as γ-globin expression was activated during erythroid differentiation of primary baboon pre-switch fetal liver cells and, to a lesser extent, of adult baboon bone marrow (BM) cells (Singh et al Exp Hematol 35:48-55, 2007). The mechanism responsible for DNA demethylation of the γ-globin promoter during erythroid differentiation remains unknown. Recent studies have shown that DNA demethylation in the early embryo is mediated by the “sixth base” 5-hydroxymethylcytosine (5-hmC) whose formation from 5-methylcytosine is catalyzed by the enzymatic activity of the three TET proteins. To investigate the hypothesis that 5-hmC mediates DNA demethylation of the γ-globin promoter during erythroid differentiation, levels of 5-hmC at Msp I (CCGG) sites within the γ- and ε-globin promoters and γ-globin IVS II region in DNA isolated from peripheral WBC, purified terminal erythroid precursors, and FACS-purified adult bone marrow subpopulations enriched for different stages of differentiation (CD117+ CD36-, CD117+ CD36+, and CD117-CD36+), were analyzed using a T4 glucosylase-MspI quantitative real time PCR assay. Levels of 5-hmC associated with the γ-globin promoter MspI site were 11.6 fold higher (p<.0001) in terminal erythroblasts (n=9) compared to peripheral blood WBC (n=4) while 5-hmC levels associated with the ε-globin promoter were 11.8 fold higher (p<.0001) in terminal erythroid precursors (n=13) compared to peripheral WBC (n=4). Levels of 5-hmC associated with the γ-globin promoter (n=9) were 9.4 fold higher (p<.0001) than with the IVS II region of the γ-globin gene (n=8) in terminal erythroid precursors suggesting that elevated levels of 5-hmC within the β-globin gene complex in terminal erythroid precursors may be localized to promoter regions. Genomic 5-hmC levels, analyzed by HPLC-MS, were similar in WBC and terminal erythroid precursors. Within purified BM subpopulations enriched for different stages of erythroid differentiation, levels of 5-hmC associated with the γ-globin promoter were 3.2 to 4.1 fold higher in the CD117+CD36+ subpopulation enriched in erythroid colony forming cells (n=4) than in CD117+CD36- (n=3; p<.01), more differentiated CD117-CD36+ (n=3; p<.02), and terminal erythroid precursor (p<.001) subpopulations. Similar differences in levels of 5-hmC associated with the ε-globin promoter were also observed between these subpopulations. Enrichment of 5-hmC within the β-globin locus in the CD117+CD36+ and terminal erythroid precursors compared to peripheral WBC was confirmed by 5-hmC affinity selection and PCR analysis. In addition, similar differences in genomic 5-hmC levels between these subpopulations were also observed by HPLC-MS analysis. Bisulfite sequence analysis showed that the changes in 5-hmC levels at the γ-globin promoter were temporally associated with loss of DNA methylation within the γ-globin promoter as erythroid differentiation progressed. TET gene expression analysis showed that TET2 expression was 3 fold less (p<.01) while TET3 expression was >7 fold higher (p<.05) in terminal erythroid precursors compared to the CD117+CD36- subpopulation. These results strongly suggest that differences in 5-hmC associated with the ε- and γ-globin promoters are the result of wider dynamic alterations of genomic 5-hmC levels during erythroid differentiation that may be mediated by differences in TET gene expression and support the hypothesis that 5-hmC is involved in the mechanism responsible for DNA demethylation of the γ-globin promoter during erythroid differentiation. Disclosures:Godley:Celgene: Research Funding.

Deep Digital Sequencing Identifies an AML Subclone with Enhanced in Vitro and in Vivo Growth Properties Associated with Disease Relapse

AbstractAbstract 407Acute myeloid leukemia (AML) is a biologically heterogenous malignancy of hematopoietic cells. All AML samples are comprised of a founding clone and usually one or more subclones that are derived from the founding clone; subclones can gain or lose mutations as they evolve from the founding clone, and often become dominant at relapse1. The clonal architecture of an AML sample can be identified using single nucleotide variants (SNVs) that cluster according to discrete variant allele frequencies (VAFs). To accurately identify clusters with common VAFs, deep digital sequencing must be performed using all of the SNVs present in each genome (hundreds of events). In this report, we studied the subclonal architecture of AML samples from UPN 452198, collected from a 55-year old woman with normal karyotype acute monocytic leukemia (FAB M5) with a high peripheral WBC count (72,700/mm3) at presentation. This bone marrow sample contained a founding clone and 3 subclones at presentation. The SNVs of the founding clone had a mean VAF of 46.4% (i.e. heterozygous mutations found in 92.8% of the cells in the bone marrow sample), including DNMT3A R882H and NPM1 W288 frameshift mutations. The mean VAFs of Subclones 1, 2 and 3 were 31.2%, 12.0% and 2.4%, respectively, and they contained all of the variants in the founding clone, along with additional variants, most notably FLT3 D835H and IDH1 R132H mutations in Subclone 1. The tumor at relapse consisted entirely of Subclone 3, which also contained 42 relapse-specific variants. We designed an oligonucleotide capture reagent to track all 118 de novo and relapse-specific variants, and obtained deep read counts (mean coverage per site: 412 reads/SNV) on the de novo AML sample under different experimental and biological conditions, as follows: 1) We showed that peripheral blood and bone marrow leukemia samples obtained at the same time had nearly identical clonal architectures. We verified this correlation using 4 additional AML samples, suggesting that clonal architecture is preserved in the peripheral blood for many AML samples. 2) We flow-sorted the leukemic peripheral blood sample into blasts, monocytes, and lymphocytes based on side-scatter characteristics and expression of CD45 and CD33. The founding clone and all three subclones were detected in the monocyte population, which was the predominant leukemic cell population in the peripheral blood. By flow cytometry, blasts comprised only 3.3% of the cells, but were strongly enriched for variants in Subclone 3 (mean VAF in sorted blasts 33.9% versus 3.0% in unsorted peripheral blood, p<0.001). Purified lymphocytes, in contrast, contained no leukemia-specific variants, implying that the founding clone for this sample did not contribute to lymphopoiesis. 3) We tested the growth properties of subclones in the de novo sample in vitro and in vivo. We injected 1 million cells from the de novo AML sample into 3 immunodeficient NSG mice, and harvested human AML blasts (co-expression of human CD45 and CD33) 14 weeks later. Although two mice engrafted with the founding clone and Subclone 1 (which comprised the vast majority of the cells in the sample), the 3rd mouse had a tumor composed entirely of the relapse-specific Subclone 3 (which accounted for only 2.4% of the variants in the de novo sample), suggesting that this subclone had a significant growth or engraftment advantage in vivo. In support of this observation, de novo AML cells were strongly enriched for Subclone 3 when grown in the presence of hematopoietic cytokines (SCF, IL3, IL6, TPO and FLT3L) for 7 days on HS27 stroma (VAF at day 7–19.7%; p<0.001) or MS5 stroma (VAF at day 7–22.8%; p<0.001), implying that this clone also had a strong in vitro growth advantage. In summary, a small subclone of AML cells at presentation — that was known to eventually contribute to relapse — had unique growth properties that were revealed using deep digital sequencing of all variants. This approach has allowed us to dissect the evolutionary history of AML clones, and to define their relationship to other hematopoietic cells in a given sample. Similar studies on additional AML samples should allow us to define the mutational profiles of subclones that are destined to contribute to relapse. This data will be essential for improving therapeutic approaches for AML patients. Disclosures:Ley:Washington University: Patents & Royalties.

FLT3 Mutation Predicts Worse Clinical Outcome of Relapsed Acute Myeloid Leukemia (rAML) Patients (Pts): Mayo Clinic Experience.

AbstractAbstract 2525 Background:Patients with relapsed AML have a very poor outcome. The molecular signature at diagnosis has emerged as a significant marker for prognostic, and possibly therapeutic, values in newly-diagnosed AML. FLT3 mutations are found in about 25–30% of AML pts. In the absence of FDA-approved agents for FLT3, the prognostic value of FLT3 mutation on clinical outcome for relapsed AML (rAML) is not well documented. Aim:To study clinical implications of FLT3 status on rAML. Methods:All adult pts with AML diagnosed and treated at Mayo Clinic between 2003–2011were studied. Retrospective data collection included pts’ demographics, laboratory tests, bone marrow biopsies, FLT3 mutation (internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutation), and survival information. IRB approval was obtained in accordance with Helsinki declaration. Chi-Square and T-test were used for comparatives between groups and Kaplan-Meier test was applied for survival estimates using JMP 9.0 software. Results:A total of 192 pts with newly-diagnosed with AML were identified. Sixty percent (115) were males, 88% (168) were Caucasians, 26% (49) had antecedent hematological disease (AHD), and 13% (25) had previous exposure to chemo- or radiotherapy. Forty-seven percent had a normal chromosomal analysis, and 18% (35) had FLT3 mutation (28 ITD, 7 TKD). In FLT3-mutated pts, median WBC was 70 x109/L (p=0.0016), hemoglobin 9.5 g /dL, and platelets 48 x109/L, compared to 8.8 x109/L, 9.1 g/dL, 50 x109/L in the rest of pts, respectively. Peripheral blood blasts were elevated in FLT3-mutated group compared to rest of pts (p 0.0059).A total of 128 pts achieved complete remission (CR), with 79 (62%) subsequently relapsed. Of the relapsed patients, 93% (74) were Caucasians, 54% (43) males, 22% (17) had AHD, and 8% (6) had prior chemo- or radiotherapy. Cytogenetics were diploid in 49% (39) and complex in 15% (12). Hematopoietic stem cell transplantation was performed in 30% (24) of pts. Eleven of 79 (14%) pts were FLT3-mutated at relapse (Group 1); with 1 pt having TKD-mutation. Forty-eight percent (38) of pts had unmutated-FLT3 (Group 2), and 38% (30) pts were FLT3-unknown (Group 3).In group 1, median WBC was 9.9 x109/L, hemoglobin 10.55 g /dL, platelets 55 x109/L, compared to 3.2 x109/L (p=0.0034), 10.55 g/dL, and 58 x109/L in group 2, respectively. Peripheral blasts were significantly elevated in group 1 compared to group 2 (38% vs 4%, p <0.0001). Surprisingly, 3/11 pts (27%) with FLT3 mutation at relapse were unmutated at diagnosis. CR was achieved in 2/11 (18%) pts in group 1 compared to 15/38 (39%) in group 2 (p=0.19). Median overall survival in rAML was significantly lower in group 1 compared to group 2 (90 days vs 229 days, p 0.0076). This survival difference was statistically more significant in pts with diploid cytogenetics at relapse compared to the rest (p 0.0009 vs 0.48, respectively). Within 1.5 years, all pts in group 1 died, compared to 21% survived in group 2. Conclusion:FLT3-mutated AML pts had a significantly higher peripheral WBC and blasts at both diagnosis and relapse. Importantly, FLT3 at relapse was found to be mutated in one quarter of pts who were wild-type FLT3 at diagnosis; raising the importance of assessing FLT3 gene on all relapses. FLT3 mutation significantly predicted worse median overall survival in rAML raising the need for novel agents, such as FLT3 inhibitors, in this population. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Bone Marrow B cell Precursor Number after Allogeneic Stem Cell Transplantation and GVHD Development

Patients without chronic graft-versus-host disease (cGVHD) have robust B cell reconstitution and are able to maintain B cell homeostasis after allogeneic hematopoietic stem cell transplantation (HSCT). To determine whether B lymphopoiesis differs before cGVHD develops, we examined bone marrow (BM) biopsies for terminal deoxynucleotidyl transferase (TdT) and PAX5 immunostaining early post-HSCT at day 30 when all patients have been shown to have high B cell activating factor (BAFF) levels. We found significantly greater numbers of BM B cell precursors in patients who did not develop cGVHD compared with those who developed cGVHD (median = 44 vs 2 cells/high powered field [hpf]; respectively; P < .001). Importantly, a significant increase in precursor B cells was maintained when patients receiving high-dose steroid therapy were excluded (median = 49 vs 20 cells/hpf; P = .017). Thus, we demonstrate the association of BM B cell production capacity in human GVHD development. Increased BM precursor B cell number may serve to predict good clinical outcome after HSCT.