Peripheral WBC Research Articles

Effects of phototherapy on Cytokines′ levels and white blood cells in term neonate with hyperbilirubinemia

Objective:Phototherapy is the most common treatment used for severe jaundice. There is increasing evidence that phototherapy can directly affect the expression and function of cell surface receptors including adhesion molecules, cytokines, and growth factor receptors. The aim of this study is to investigate the effect of phototherapy use on the levels of interleukin (IL)-1α, IL-6, and tumor necrosis factor (TNF)-α as cytokine expressions from keratinocytes, and also white blood cell counts in the treatment of neonate with hyperbilirubinemia.Materials and Methods:We studied 32 term newborns with hyperbilirubinemia. Blood samples were obtained before and 72 h after phototherapy. Serum levels of IL-1α, IL-6, TNF-α, and WBC count were measured in the samples using appropriate methods.Results:Serum TNF-α at 72 h of exposure to phototherapy increased, while the levels of IL-1α and IL-6 at the same time were decreased. These changes were not statistically significant. WBC counts rose significantly with phototherapy.Conclusion:Phototherapy in term neonate does not affect cytokines’ levels, but can raise peripheral WBC count.

Clinical Study on Effect of Yanshu Injection in Efficacy Enhancing and Toxicity Reducing of Chemotherapy in Patients with Malignant Tumor

Objective To observe the effect of Yanshu Injection on the efficacy enhancing and toxicity reducing for the chemotherapy in patients with malignant tumor.Methods Eighty-three malignant tumor patients were randomly divided into treated group and control group.In their therapeutic period of chemotherapy,Yanshu Injection was given additionally to the treated group(43 patients),by intravenous dripping of normal saline once per day for 7d as one course.The control group(40 patients)was given conventional chemotherapy.Results Compared with the control group,the treated group showed a low progressive incidence,less decrease of peripheral WBC and platelet count(P0.05).Conclusion Yanshu Injection combined with chemotherapy is effective in improving the therapeutic efficacy,elevating the immune function of organism and lessening the toxicity of medicine.

Quantitative Analysis of Tissue and Developmental Stage-Specific Differences in DNA Methylation throughout the β-Globin Gene Locus by DNA MassARRAY,

Abstract 3196DNA methylation plays a fundamental role in developmental regulation of γ-globin gene expression. The baboon is an excellent animal model to study the role of DNA methylation in developmental globin gene regulation because 1) the sequence and arrangement of genes within the β-globin locus are highly conserved between baboon and man, 2) only simian primates exhibit true fetal-stage γ-globin expression, 3) large numbers of highly purified erythroid cells can be isolated from baboon fetuses derived from timed pregnancies at varying stages of gestation and from the bone marrows of adult baboons, and 4) treatment of baboons with DNA methyltransferase inhibitors induce abundant γ-globin expression in adult baboons. Bisulfite sequence analysis data has shown that DNA hypomethylation within the 5’ promoter regions of the ε- and γ-globin genes is associated with expression during the embryonic and fetal stages of development (Lavelle et al Blood Cell Mol Dis 36:269, 2006), and developmentally regulated, large extended domains of DNA hypomethylation surrounding expressed genes have been also reported (Lathrop et al Exp Hematol 37:807, 2009). Although analysis of DNA methylation by bisulfite sequence analysis is considered to be the “gold standard”, quantitative analysis by this method is laborious and limited by the ability to perform DNA sequence analysis of large numbers of clones isolated following PCR amplification and bisulfite modification. Recently, a new quantitative method, MassARRAY, has been developed that allows direct quantitative analysis of cytosine methylation within defined PCR amplicons, thus eliminating the need for the laborious sequence analysis of large numbers of clones and allowing more precise quantitative analysis. To develop MassARRAY technology as a tool for studies of DNA methylation of the β-globin gene locus we compared the ability of MassARRAY and bisulfite sequencing to quantitate differences in DNA methylation at 13 specific CpG sites spanning the baboon β-globin gene cluster from HS4 within the LCR to the 5’ β-globin gene region in peripheral WBC, primitive nucleated erythrocytes isolated from a baboon fetus at 40 d gestation, purified erythroid precursors from pre-switch fetal liver, post-switch fetal bone marrow (BM) and adult BM pre- and post-treatment with decitabine administered by either subcutaneous injection (0.5mg/ml sc; 10d) or orally (0.25mg/kg/2d per week; 8 weeks) in combination with tetrahydrouridine. PCR amplification of specific regions of the baboon β-globin gene locus from bisulfite-converted DNA was performed using primers containing the 5’ T7 and 10mer tags selected using Sequenom Epidesigner software. PCR conditions were optimized to yield single bands by agarose gel analysis in all reactions. PCR products were treated with shrimp alkaline phosphatase and in vitro transcription and RNase digestion performed, followed by analysis using the Maldi-TOF MassARRAY system (Sequenom). Quantitation of DNA methylation of specific CpG residues was performed using EpiTYPER software. To compare results obtained by MassARRAY and traditional bisulfite sequence analysis, PCR products from bisulfite-converted DNA were also cloned in the PCR4 vector and sequence analysis of at least 10 clones performed for each amplicon. Results of MassARRAY and bisulfite sequence analysis were in close agreement. Analysis of all sites in all samples yielded a mean difference of 12.3 + 9.2% dmC between these two techniques. Four CpG sites within the LCR region were highly methylated in WBC DNA compared to erythroid tissues. Developmental differences in DNA methylation in the 5’ ε- and γ-globin regions were associated with stage-specific expression of these genes. Modest differences (10–15% dmC) in DNA methylation of four CpG sites in the 5’ γ-globin gene region in BM erythroid cells isolated from baboons treated with oral low-dose decitabine-tetrahydrouridine in combination detected by DNA MassARRAY were consistent with the modest 15–20% elevations of fetal hemoglobin (HbF). In conclusion, quantitative analysis of DNA methylation by MassARRAY technology provides an alternative, less laborious, sensitive quantitative methodology for analysis of differences in DNA methylation throughout the β-globin gene locus during development and following treatment with HbF-inducing agents. Disclosures:No relevant conflicts of interest to declare.

The CXCR4 Antagonist AMD 3100 Induces Rapid Mobilization of Facilitating Cells and Hematopoietic Stem Cells in Mice

Abstract 4694Bone marrow transplantation (BMT) offers great promise for treating red blood cell disorders, inherited disorders of metabolism, autoimmune diseases, and inducing donor-specific tolerance to organ transplants. However, the widespread application of this approach is dependent upon the development of less toxic strategies for BMT and avoidance of graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FC) facilitate engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex barriers without causing GVHD. We previously reported that Flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) synergistically mobilize FC and HSC into the peripheral blood (PB). Recently, AMD 3100 has been found to be a rapid mobilizing agent whose effect occurs within hours after injection. It is a macrocyclic compound and potential fusion inhibitor that antagonizes CXCR4 alpha-chemokine receptor for its effect on HSC mobilization. CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1), are important in HSC homing and maintenance in the bone marrow microenvironment. Here, we investigated the effects of AMD 3100 on the mobilization of FC and HSC into PB in combination with FL and G-CSF.A dose titration of AMD 3100 was first performed. B6 mice were injected subcutaneously with AMD 3100 with the doses ranging from 1.25 mg/kg to 10 mg/kg. PB was obtained 0.5, 1, 3, and 6 hours post-injection. After individual count of peripheral blood mononuclear cells (PBMC), cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−). The numbers of PBMC significantly increased even 0.5 hour after AMD 3100 treatment and peaked at 1 h. The maximal mobilization of PBMC was noted at 1 h with 5.0 mg/kg AMD 3100. Treatment with 5.0 mg/kg AMD 3100 caused a 3.1-fold increase of WBC at 1h compared with saline treated controls. An increase of FC was detectable with all doses of AMD 3100. The numbers of FC peaked between 1 and 3 h, and declined rapidly to resemble saline-treated controls at 6 h after. A 5.9-fold increase of FC was observed at 1 h with 5.0 mg/kg AMD 3100 (P = 0.012). These data suggest that AMD 3100 is a potent cell mobilizer from bone marrow to PB.We next investigated the effect of AMD 3100 in combination with FL and G-CSF on the mobilization of FC and HSC into PB. B6 mice were injected with FL (day 1 to 10), G-CSF (day 4 to 10), and AMD 3100 (day 10). PB was obtained 1 h after injection on day 10. After performing a count of peripheral WBC, cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−) and HSC (Lin−/Sca-1+/c-kit+) mobilization. The maximal mobilization of PBMC was observed when animals were treated with AMD 3100/FL/G-CSF. The numbers of PBMC with AMD3100/FL/G-CSF treatment increased with 17.2-fold and 6.4-fold when compared with controls treated with saline or AMD 3100 alone (P < 0.00001), respectively. A maximal elevation of both FC and HSC was detected when AMD 3100 was added to FL/G-CSF treatment and reached 1.91 ± 0.42 × 103/μl (Figure 1A) and 1.89 ± 0.35 × 103/μl (Figure 1B), respectively. The increase of FC and HSC was significant. There was a 10.1-fold increase in FC and 230.8-fold increase in HSC when compared with recipients treated with AMD 3100 alone (P < 0.00001). AMD 3100/FL/G-CSF treatment resulted in a 1.7-fold of FC and 2.2-fold increase of HSC when compared with recipients treated with FL/G-CSF (P < 0.05). In summary, AMD 3100, FL, and G-CSF show a highly significant synergy on the mobilization of FC and HSC. This study may be clinically relevant in efforts to mobilize immunomodulatory FC and HSC to PB for transplantation, especially to induce tolerance for organ transplant recipients. Disclosures:Ildstad:Regenerex, LLC: Equity Ownership. [Display omitted]

Young Adults Presenting with Extramedullary Acute Myeloid Leukemia Have A Unique Sensitivity to High Doses of Anthracyclines: Subset Analysis of ECOG 1900,

Abstract 3619Extramedullary (EM) acute myeloid leukemia (AML) is generally associated with a poor prognosis. We evaluated the baseline characteristics, response and survival characteristics in a subset of patients age ≤ 60 years with de novo AML and EM manifestations at presentation, treated on the Eastern Cooperative Oncology Group E1900 protocol: daunorubicin 45 or 90 mg /m2 x3 days in conjunction with cytarabine 100 mg/m2 × 7 days followed by risk-stratified consolidation. (Fernandez et al New Engl J Med 2009) Methods:Patient baseline characteristics were compared using Fisher's exact test if they were categories and Wilcoxon rank sum tests if they were continuous. Logistic regression models were used for complete remission (CR) rates and Cox regression models were used for overall survival (OS) and disease-free survival (DFS). The univariate models included indication of whether patient had extramedullary disease involvement, adjusted by induction treatment. The multivariate models were further adjusted for age, gender, cytogenetic risk, ECOG PS, WBC, platelets, marrow blast, hemoglobin, peripheral blasts, and secondary AML. All P values were based on 2-sided tests. Results:58 (8.9%) of 651 patients in the study presented with evidence of EM at diagnosis. Patients with EM disease were younger and presented with higher peripheral WBC counts and circulating blasts than those without. Immunophenotyping did not demonstrate a difference in baseline CD56 expression (p=0.77). Sites of EM involvement included gingival hypertrophy (n= 33), skin (n= 7), central nervous system (n= 6), lymphadenopathy (n=6), mediastinum (n=2) and other (n=6). 3 patients had multiple sites. Cytogenetic risk in the EM subgroup was favorable (8.6%), indeterminate (inadequate/missing) (34.5%), intermediate (37.9%) and unfavorable (19%) and did not differ statistically from those without EM disease.The complete remission (CR) rates were similar for patients with EM disease compared to those patient's without, regardless of the anthracycline dose (univariate model p= 0.93; multivariate model p=0.88. DFS (patients in CR) and OS of patients with EM disease was not significantly different in the univariate (p= 0.99, p=0.49) or multivariate models (p= 0.92, p=0.37), respectively. On multivariate modeling patients with EM AML who received the standard-dose of daunorubicin had a shorter OS (HR 1.86, 95% CI 1.20 to 2.91, p=0.006). OS was not improved in EM patients treated with high-dose daunorubicin (HR 0.62, 95% CI 0.34 to 1.14, p=0.12) (Table). There was a significant difference in OS for patients with EM treated with the 90 mg/m2 dose of daunorubicin compared to patients with EM treated with the 45 mg/m2 dose (multivariate model: HR 0.30, 95% CI 0.12 to 0.76, p=0.01). Conclusion:Younger patients with de novo EM AML are sensitive to higher doses of anthracycline in induction therapy and should be treated in a similar fashion as patients without EM involvement. For both groups the higher doses of anthracycline are recommended as CR rates, DFS and OS are improved. Further evaluation of immunophenotypic and molecular/ genetic characteristics should be undertaken in this subset of patients. Disclosures:No relevant conflicts of interest to declare.

Cerebrospinal fluid metabolomics in persons with PSEN1 mutations

WBC counts held when the comparisons were made among persons from families with APP or PSEN1 mutations alone though the differences were not significant. There was no clear effect of age or disease stage on WBC count. Conclusions: Though preliminary, our results suggest that FAD MCs have lower peripheral WBC counts than their non-mutation carrying kin. Though lower levels in PSEN1 MCs might be explained by aberrant processing of Notch, the explanation of lower levels in APP MCs is less clear though APP expression is seen in the thymus and lymph nodes.

FLT3-positive acute myeloid leukemia: Does timed sequential induction therapy influence outcome?

6544 Background: Mutations of FLT3, ITD, or TKD, occur in 30% of AML, making them the most common in AML. FLT3-ITD has been correlated with higher WBC counts and bone marrow (BM) blast counts and decreased disease-free survival (DFS) and overall survival (OS). Median OS has been reported as 2.8-6.4 months (mo) in FLT3-ITD+ vs. 10-14.9 mo in FLT3-ITD wild-type (WT). To date, no treatment regimen has been shown to impact the outcome of FLT3+ patients (pts). Various studies have shown that type and intensity of therapy or use of a reinforced induction regimen could influence the outcome of FLT3-ITD+ pts. We retrospectively studied the outcome of pts with FLT3+ AML treated with a regimen based on timed sequential therapy concept. Methods: 33 pts were treated with 2 doses of cytarabine 2 gm/m2 IVPB 12 hours apart followed by 1 dose of mitoxantrone 30 mg/m2 IVPB on days 1 and 5. For pts ≥ 70 years, cytarabine was reduced to 1.5 gm/m2. Study end points were overall response rate (ORR), DFS, and OS. Results: 19 pts were FLT3-WT, 9 were FLT3-ITD+ and 5 were FLT3-TKD+. Median age was 50 years in the FLT3-WT group (30-73), 50 in the ITD+ group (30-72) and 51 in the TKD+ group (36-66). Cytogenetics, NPM status, peripheral WBC count, Peripheral Blast% & BM Blast% are summarized in the table. ORR (CR + CRi) was 78.9% in the FLT3-WT group and 100% in both FLT3+ groups. Median DFS for FLT3-WT pts was 7.1 mo (0.4 – 17.6), 9.9 mo in the ITD group (0.9-33.7) and 6.6 mo in the TKD group (2.3-14.8). Median OS for FLT3-WT pts was 8.7 mo (0.6 – 57.9), 14.2 mo in the ITD group (2.2-41.5) & 8 mo in the TKD group (3.3-18.7). Conclusions: This regimen was highly effective in achieving remission in FLT3-ITD+ AML resulting in improved survival. The lack of unfavorable cytogenetics in this group may have influenced the outcome. This regimen appears to be promising for FLT3+ pts, and further studies are needed. All Pts FLT3-WT FLT3-ITD FLT3-TKD Median age 50 50 50 51 Favorable cytogenetics 7 4 1 2 Intermediate cytogenetics 19 9 8 2 Unfavorable cytogenetics 7 6 0 1 NPM (# positive/# tested) 7 / 13 2 / 5 5 / 6 0 / 2 Median peripheral WBC # 20.19 17.14 49.27 74.02 Median peripheral blast% 23 3 59 47.55 Median BM aspirate blast% 68.5 68 85 37.5 ORR 87.9% 78.9% 100% 100% Median DFS (mo) 7.1 7.1 9.9 6.6 Median OS (mo) 9.8 8.7 14.2 8

Absence of leukocytosis in bacteraemic pneumococcal pneumonia

Evaluation of patients with respiratory symptoms in primary care medicine is often based on peripheral WBC count that dictates the extent of diagnostic investigation. A normal WBC count may result in a limited investigation, often omitting chest radiography. To determine the extent to which patients hospitalised with bacteraemic pneumococcal pneumonia have no leukocytosis at presentation. A retrospective analysis was performed of patients with bacteraemic community-acquired pneumococcal pneumonia from 2000 to 2007 in a community care academic medical centre. Records were reviewed for symptoms, signs, and laboratory data including pneumococcal serotypes, chest radiographs on admission, and outcome. 21% of the patients presented with a normal WBC count (16.7% of the children and 25.6% of the adults). Among this population with a normal WBC count at presentation, 90% of the adults and 70% of the children developed leukocytosis within a few days after admission. In this study, in as many as one-fifth of all the patients with bacteraemic pneumococcal pneumonia, there was no leukocytosis at presentation. We therefore suggest that every patient with clinically suspected pneumonia should undergo chest radiography even if the WBC count is normal.

Evaluation of monocyte-derived dendritic cells, T regulatory and Th17 cells in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors

Immunotherapy with dendritic cells (DC) may constitute a new and advantageous option for patients with chronic myeloid leukemia (CML) who respond to therapy with tyrosine kinase inhibitors (TKI), but do not reach complete cytogenetic or molecular remission. In this study, we evaluated the immunophenotype of DC generated from monocytes (Mo-DC) of patients with CML and the influence of TKI therapy on the results of CML-DC generation. We also measured the percentages of T regulatory cells (Tregs) as well as Th17 cells in 19 untreated patients suffering from CML, and in 28 CML patients treated with TKI. We found that DC can be reliably generated from the peripheral blood CD14+ cells of untreated CML patients. But we observed a persistent expression of CD14 monocyte marker on DC from CML patients, together with lower percentages of Mo-DC with expression of CD1a (p = 0.002), CD80 (p = 0.0005), CD83 (p = 0.0004), and CD209 (p = 0.02) compared to healthy donors. There was an adverse correlation between WBC count and the percentage of Mo-DC with co-expression of CD80 and CD86 (R = -0.63; p = 0.03). In patients treated with TKI, we observed higher efficacy of DC generation in seven-day cultures, compared to untreated patients. Expression of CD209 on DC was higher in patients treated with TKI (0.02). The duration of TKI therapy correlated adversely with MFI for CD1a (R = -0.49; p = 0.006) and positively with MFI for CD83 (R = 0.63; p = 0.01). Percentages of CD4+CD25highFoxP3+ cells (p = 0.0002) and Th17 cells (p = 0.02) were significantly higher in untreated CML patients compared to healthy controls. There was a significant correlation between the percentage of Treg cells and the percentage of peripheral blood basophiles (R = 0.821; p = 0.02). There were no changes in Tregs or Th17 cell percentages in CML patients after six months of TKI therapy. However, the expression of intracellular IL-17 in Th17 cells correlated negatively with the time of TKI therapy in the whole group of treated patients (R = -0.516; p = 0.04). We noted a correlation between IL-6 serum level and peripheral blood WBC count (R = 0.492; p = 0.04). There was also an inverse correlation between the serum level of IL-6 and the duration of TKI therapy (R = -0.66; p = 0.03). Taken together, our data shows that mature DC can be generated from CML patients treated with TKI, and that the yield of Mo-DC is higher in patients treated with TKI than in patients with active disease. This should encourage further trials with DC immunotherapy in patients with cytogenetic response after TKI therapy. We also found increased frequencies of T regulatory and Th17 cells in CML patients, which might suggest their potential role in immunity against this disease. Further studies are needed to determine if manipulation of these cell populations might improve the results of DC immunotherapy.

Abstract 5444: Real-time RT-PCR of terminal deoxynucleotidyl transferase (TdT) is a useful tool for identification of minimal leukemia in cerebrospinal fluid

Abstract Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. The detection of cerebrospinal fluid (CSF) infiltration by leukemic cells as performed by morphological assessment is subjective and unable to detect low levels of involvement. Therefore, newer and more accurate techniques are required to more efficiently detect minimal leukemic cell involvement. For this reason, a quantitative, Real-time RT-PCR targeting the TdT molecule was developed and tested to allow an objective and sensitive methodology for detecting CNS leukemia. 35 diagnostic CSF samples were subjected to the qRT-PCR assay for TdT. Only 4/35 samples were positive by cytomorphological detection. Of these 4 samples, one sample was diagnosed with CNS disease (CNS 3) and three samples had lower numbers of leukemic blasts (CNS 2). When compared to morphology results, as the gold standard, we found 24 out of the 35 samples to be positive by qRT-PCR. 20 CSF samples that were negative by morphology had detectable TdT by RT-PCR, possibly indicating leukemic infiltration below the level of cytomorphological detection. Serial samples for the 4 CNS-positive patients were tested by qRT-PCR and showed persistent positivity with gradual reduction in quantity of TdT with chemotherapy administration. 11 samples had no detectable TdT by RT-PCR and were found to be negative by cytomorphological assessment as well. In patients with no cytomorphologically detected blasts, TdT positivity was not associated with the level of peripheral blood WBC count. This methodology provides an objective, quantitative and sensitive tool for the detection of leukemic blasts in the CSF. This allows us to detect molecular CNS involvement in CNS negative patients. Although the outcome for all CNS negative patients is good, this has been achieved with fairly intensive prophylactic CNS-directed therapy, reduction in CNS-directed therapy may be possible in molecularly negative patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5444. doi:10.1158/1538-7445.AM2011-5444

Epidemiology and risk factors for non-Candida albicanscandidemia in non-neutropenic patients at a Chinese teaching hospital

The purpose of this study was to describe the epidemiology of nosocomial candidemia and identify risk factors involved in infections caused by non-C. albicans Candida species in a Chinese tertiary care center over a 10-year period. A total of 102 cases of nosocomial candidemia in non-neutropenic patients admitted from 1998 through 2007 were included in the study. Candida albicans remained the most common causative agent, accounting for 57.8% of all cases, followed by C. tropicalis (12.8%), C. parapsilosis (10.8%) and C. glabrata (10.8%). Comparison of C. albicans and non-C. albicans candidemia by multivariate logistic regression showed that factors independently associated with non-C. albicans candidemia included head trauma (OR, 5.34; 95% CI, 1.18-24.17; P = 0.029) and bacterial sepsis (OR, 3.58; 95% CI, 1.17-10.98; P = 0.026). Factors independently associated with C. albicans candidemia included tracheal intubation (OR, 0.26; 95% CI, 0.08-0.92; P = 0.037), and increased peripheral WBC count (OR, 0.84; 95% CI, 0.74-0.95; P = 0.006).

Longitudinal modulation of immune system cytokine profile during pregnancy

Objective To characterize immune modulation as expressed by cytokine assays at three time-points in human pregnancy. Study design This is a prospective, longitudinal study of a broad panel of cytokine expression during singleton pregnancies resulting in an uncomplicated, full-term, live births. Peripheral blood was obtained at 8–14, 18–22, and 28–32 weeks gestation. Six cytokines – IFN-γ, IL-4, TNF-α, IL-1β, IL-6, and IL-10 – were measured in supernatants obtained from whole blood stimulations with PHA or LPS and were compared to unstimulated controls. Samples were processed by Luminex-100 MAP®. We used Generalized Linear Models (GLM) to evaluate cytokine trajectories. Results Complete data were obtained for 45 uncomplicated pregnancies. Overall, peripheral blood WBC’s demonstrated dampened cytokine responses. However, over the course of pregnancy, we found enhanced counter-regulatory cytokine expression (e.g., shown by increased IL-10). Conclusion The overall decrease in pro-inflammatory cytokines and increase in counter-regulatory cytokines as uncomplicated pregnancy progresses supports the evolving concepts of immunoregulation for the maintenance of a viable pregnancy.

Improved Survival and Recovery of Hematopoiesis Following Lethal Radiation by Chloroquine-Mediated Activation of ATM

AbstractAbstract 2228Irreversible bone marrow damage and impaired blood formation is the primary cause of death following exposure to high doses of radiation. Moreover, the rate at which radiation is delivered may have a profound impact on cytotoxicity; prolonged exposure at a low dose-rate (LDR; 9.4 cGy/hr) has been found to induce greater cell death than the same total dose given at a high dose-rate (HDR; 4500 cGy/hr). Few non-toxic agents are presently available that can offer substantial protection against radiation induced bone marrow failure and death, especially during LDR exposure. We previously demonstrated that chloroquine, a commonly used agent in the treatment of malaria and rheumatologic diseases, can prevent LDR radiation induced cytotoxicity of cell lines in vitro and studied its effects on hematopoiesis in vivo. We initially quantified the effects of LDR radiation on C57/B6 mice and found that 9 Gy delivered at 9.4 cGy/hr for 95.7 hrs induced death in 13/19 (68%) of animals at 15–35 days after radiation. The administration of syngeneic bone marrow cells (1 × 106 cells) immediately after LDR radiation completely rescued animals (10/10) demonstrating that bone marrow failure was responsible for LDR radiation induced death similar to HDR radiation. Next we treated mice with chloroquine (0.0594 mg/17g body weight, i.p.) 24 hrs and 4 hrs prior to exposure to LDR radiation and found that it significantly improved survival (80%, p < 0.05) compared to untreated animals exposed to LDR radiation (32%). We examined hematopoietic recovery following LDR radiation and found that the peripheral WBC was significantly greater in mice treated receiving chloroquine (3.4 × 106/ml vs 1.1 × 106/ml at day 16, p<0.05). Similarly, we found that in vivo chloroquine treatment significantly increased the recovery of bone marrow myeloid CFC (p=0.02), suggesting that it impacted myeloid progenitors. To further validate this finding, we transplanted bone marrow from LDR irradiated mice into lethally irradiated CD45 congenic recipient mice, and found a significant improvement in early engraftment (4.2% vs. 0.4% engraftment at 6 weeks post-transplant, p=0.015). Chloroquine has been found to protect cancer cell lines from LDR radiation in vitro by activating ATM, an essential DNA damage sensor. We examined the effect of chloroquine on ATM and treated unradiated lin- bone marrow cells with chloroquine in vitro (35 ug/ml, 2 hr). Compared to control cells, chloroquine treated cells expressed 2.5-fold more phosphorylated ATM suggesting that the activation of ATM by chloroquine abrogated the lethal effects of LDR radiation in hematopoietic progenitors. We confirmed that ATM was required for chloroquine-mediated radioprotection by studying ATM null mice. In contrast to wild type mice, chloroquine treatment failed to protect ATM null mice from LDR radiation (9 Gy total) with 8/13 (62%) and 9/13 (69%) of animals surviving in treated or non treated mice, respectively (p=0.86). These data suggest that chloroquine exerts a radioprotective effect from LDR radiation by activating ATM in vivo, and may represent a novel means of limiting acute bone marrow failure in the event of widespread environmental LDR radiation exposure. Disclosures:Matsui: Pfizer: Consultancy; Bristol-Meyers Squibb: Consultancy; Infinity Phamaceuticals: Consultancy, Patents & Royalties; Merck: Consultancy, Research Funding; Geron Corporation: Research Funding.

Combined Low-Dose Homoharringtonine, Cytarabine and Granulocyte Colony-Stimulating Factor for Patients with Advanced Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia Transformed From MDS

AbstractAbstract 4004A pilot study of the efficacy and toxicity of the low-dose cytarabine and homoharringtonine in combination with granulocyte colony-stimulating factor (G-CSF) (CHG protocol) in patients with advanced myelodysplastic syndromes (MDS) or MDS-transformed acute myeloid leukaemia (t-AML) was conducted. A total of 50 patients (31, advanced MDS; 19, t-AML) were enrolled in this study. All patients were administered the CHG regimen comprising low-dose cytarabine (25 mg/d, intravenous continuous infusion, days 1 C14), homoharringtonine (1 mg/d, intravenous continuous infusion, days 1 C14) and G-CSF (300 μg/d, subcutaneous injection, days 0 C14, intermitted when the peripheral WBC count reached >20 °Á 109/L). This regimen was followed by conventional chemotherapy as post-remission therapy when complete remission (CR) was achieved. The overall response rate was 68.0% after one course of the CHG regimen was applied. Of the total 50 patients, 24 (48.0%) achieved CR and 9 (18.0%) achieved partial remission (PR). The median overall survival (OS) was 14.1 months. Among the patients aged ≥ 70 years, the response rate was 81.0% (71.4% CR and 9.6% PR), while in patients aged < 70 years, the response rate was 55.2% (31.0% CR and 24.1% PR) (P < 0.01). There were no statistically significant differences for CR, PR and OS when the patients were grouped by blast percentage in bone marrow and karyotypes, respectively. 8 of the 24 CR patients who just received post-remission regimens of HA (homoharringtonine and cytarabine) and DA (daunorubicin and cytarabine) relapsed rapidly and just kept a mean CR of 4.3 months. Otherwise, the other 14 CR patients alternatively received succeeded chemotherapy, which combined mitoxantrone, idarubicin, pirarubicin or aclarubicin with cytarabine or decitabine. The mean CR duration of these 14 patients reached 15.5 months with 4 still maintaining continuous CR. No treatment-related deaths were observed. Myelosuppression was mild to moderate, and no severe non-haematological toxicity was observed. Thus, CHG priming regimen as an induction therapy is well tolerated and effective in advanced MDS or t-AML patients. Disclosures:No relevant conflicts of interest to declare.

Long-term survey of outcome in acute promyelocytic leukemia: a single center experience in 340 patients

The aims of this study are to investigate the outcome and prognostic factors influencing long-term survival on patients with acute promyelocytic leukemia (APL). A total of 340 APL patients admitted to the Department of Hematology from January 1988 to December 2009 were enrolled in this study. All patients received all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO) with anthracycline-based induction therapy. After three courses of consolidation chemotherapy, 279 patients received 2 years of maintenance therapy. Survival analyses were carried out using the Kaplan-Meier method and the Cox regression model. In total, 288 achieved CR with the CR rate of 84.7%, and 50 patients died during induction therapy. Univariate analysis identified the following three risk factors for hemorrhagic mortality: fibrinogen level (<1.0 g/l) (P = 0.0007), initial peripheral WBC count(>4 × 10(9)/l) (P = 0.0001), as well as the presence of coagulopathy(P < 0.0001). With a median follow-up of 49 (6-255) months, the estimated 5-year overall survival (OS) and relapse-free survival (RFS) were (89.0 ± 2.4)% and (83.7 ± 2.6)%, respectively. Cox regression analysis of the 290 patients showed initial WBC count, years of diagnosis, and the status of PML-RARα in remission seemed to be independent prognostic indicators for OS and RFS (P = 0.03, P < 0.01 and P = 0.0001, respectively). Cytogenetics in addition to above three variables remained significant for RFS (P = 0.01). Our retrospective observations suggest that the combination of ATRA and/or ATO with anthracycline-based therapy may have useful implications in the perspective of long-term prognosis for adult APL.

Single nucleotide polymorphisms in DNA repair gene ERCC1 predict clinical response to platinumbased chemotherapy in non-small cell lung cancer

Objective To assessed whether single nucleotide polymorphisms (SNP) of DNA-repair genes ERCC1 predict efficacy and prognosis of non-small-cell lung cancer (NSCLC) patients treated with platinum-based chemotherapy. Methods A retrospective dataset of 117 patients with NSCLC were routinely treated with cisplatin (DDP) or carboplatin (CBP) based regimens as first-or second-line chemotherapy. The allelotyping of DNA-repair genes polymorphisms were determined via PCR-RFLP using genomic DNA obtained from peripheral WBC. Results ERCC1 (Asn118Asn) genotype was significantly associated with response to treatment. Patients with either one or two C alleles (C/C, C/T) at Asn118Asn were more likely to respond to platinum-based chemotherapy compared with those without the C allele (Odds ratio = 0.19; 95% CI: 0.041-0.872; P＜ 0.05, by binary logistic regression). There was a significant association between the ERCC1 C8092A polymorphism and OS (P＜0.05, by log-rank test), with median survival times of 9 (C/C) and 16 (C/A or A/A) months, respectively,suggesting that any copies of the A allele were associated with an improved outcome. Cox's multivariate analysis suggested that the joint effect of ERCC1 polymorphic variants (C8092A and N118N) (Odds ratio, 4. 37; 95% CI: 1.26-15. 23;P＜0.05) was independent prognostic factors for OS in advanced NSCLC patients treated with platinumbased chemotherapy. Conclusion Assessment of genetic variations of ERCC1 could predict platinum-based chemotherapy outcome in advanced NSCLC. Key words: Single nucleotide polymorphisms; DNA repair gene ERCC1; Non-small cell lung caner; Chemotherapy

Can Short Antibiotic Treatment be Widely Used in Developed Countries

Childhood pneumonia is the most common killer of children <5 years worldwide. Although in the developed world pneumonia does not commonly result in death, it is an extremely common disease. Treatment is almost universally with antibiotics. However, the lack of appropriate definition of pneumonia, the difficulty in diagnosing the pathogen and cultural differences make it extremely difficult to agree on the ideal treatment. A short antibiotic course is preferred to a longer one, since if equally effective, it should be associated with lower cost, less adverse events and lower rates of antibiotic resistance in the community. An extensive literature search using a combination of the words “pneumonia”, “community-acquired”, “treatment”, “drug”, “antibiotic”, short”, “shortened”, “day (s)”, and “child” resulted in a few articles only dealing with short duration treatment of pneumonia in children, all in the developing world and most dealing with pneumonia defined by the WHO criteria (used in locations where no modern medicine can be applied). We undertook a prospective, double blind, randomized study, comparing high-dose amoxicillin (80 mg/kg) administered for 5 vs. 10 days in children with community-acquired alveolar pneumonia with the following inclusion criteria: 1) <5 years old; 2) radiologically proven alveolar pneumonia; 3) temperature “38.5 °C; 4) peripheral WBC” 15,000/mm3. We followed the children both clinically and with laboratory findings for a month. The study will be unblinded after January 31st and the preliminary results will be presented. In conclusion: Paucity of data and difficulty in defining childhood pneumonia in the developed world result in confusion in regard to short treatment. However, the first studies will reveal, at least in some defined subgroups of pneumonia, the efficacy and the potential use of short-course treatment in childhood pneumonia. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive

A study of chest X-ray classification of farmer's lung--correlation between X-ray pattern and background factors

Patients with farmer's lung were classified into five groups according to their chest X-ray pattern, and the background of each group was studied with respect to stage, severity, and prognosis. Type I group, which has fine nodular shadows on chest X-ray, showed a significantly high percentage of lymphocytes in BAL cells (p less than 0.01). Type V group, which has irregular patchy shadows on chest X-ray, showed increased peripheral WBC counts, increased CRP score, and decreased PaO2 compared to the other groups. Type II, III, and IV groups, which are characterized by combined X-ray patterns such as nodular shadows, ring-like shadows, and irregular pulmonary markings, had no specific background features according to our study. Our results suggest that type I and type V groups are likely to represent acute phase and severe phase, respectively. We could not identify any significant factors indicative of chronic disease in terms of restrictive and diffusion impairment of pulmonary function tests and pathologic findings obtained by TBLB. The chest X-ray classification we proposed is useful for the diagnosis of the acute and severe stages of farmer's lung.

ベイズ解析を用いた診断確率定量法による MRSA 下気道感染症診断の試み

Using quantitative Bayesian analysis as a clinical epidemiological approach, we developed a diagnosis for lower respiratory tract infection (LRTI) due to Methicillin-resistant Staphylococcus aureus (MRSA). We retrospectively reviewed the charts of 181 subjects--a derivation cohort-with MRSA retrieved from lower respiratory specimens June 2006 to March 2008. Dividing them into infection or colonization (no infection) groups, we compared them for the presence or absence of clinical parameters, including fever > 38 degrees C, MRSA >106 CFU (colony-forming units)/mL, phagocytosis on Gram staining, serum albumin < 3.0 g/dL, and peripheral WBC count > 15,000/mL. We them determined positive and negative likelihood ratios (LR +, LR -) for these parameters to quantify MRSA-LRTI diagnostic probability based on combined likelihood ratios (Bayesian analysis). We then determined Bayesian MRSA-LRTI diagnostic probabilities (BDPs) in 40 subjects with respiratory MRSA--a validation cohort-from May 2008 to October 2008 clinically judged with either infection (n = 14) or colonization (n = 26) by infection control personnel (ICP) blinded to the test (parameter LR+ and LR -). BDPs (mean +/- SD) quantified by combining the four parameters-fever, MRSA CFU, phagocytosis, and serum albumin-were 62.3 +/- 25.4% for 14 judged with infection, and 40.2% +/- 20.4% for 26 patients judged with colonization (p = 0.005). Using a diagnostic probability of 51% as the cut off, we compared positive and negative predictive Bayesian diagnoses ICP judgment, i.c., 77% vs. 85%. The Bayesian approach proved useful in quantitatively diagnosing infectious disease such as MRSA-LRTI that lack established diagnostic, and may aid physicians in deciding the need for specific antimicrobial therapy.

A Prospective Study of Telomere Length Measured by Monochrome Multiplex Quantitative PCR and Risk of Non-Hodgkin Lymphoma

Telomere length plays an important role in the maintenance of chromosomal stability and in tumorigenesis. We hypothesized that telomere length in peripheral WBC DNA obtained from healthy individuals would be a predictor of future risk of developing non-Hodgkin lymphoma. Using a new assay to measure relative telomere length, monochrome multiplex quantitative PCR, which strongly correlates with telomere length measured by Southern blot (Spearman r = 0.91, P < 0.0001) and has high precision (coefficient of variation = 7%), we compared telomere length in peripheral WBC DNA in 107 incident male non-Hodgkin lymphoma cases and 107 matched controls within the prospective Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study cohort. Median (10th, 90th percentile) telomere length was 1.10 (0.79, 1.43) in cases and 1.02 (0.78, 1.26) in controls (P = 0.0017, Wilcoxon sign test). There was a strong dose-response relationship between quartiles of telomere length and risk of non-Hodgkin lymphoma overall [odds ratios (95% confidence intervals) by quartile: 1.0; 1.1 (0.4-2.7); 1.8 (0.7-4.9); and 3.6 (1.4-8.9); P trend = 0.003], and this association was similar across the most common non-Hodgkin lymphoma subtypes present in this study. These results suggest that longer telomere length may be a potential predictor for future risk of non-Hodgkin lymphoma.