Peripheral Cell Counts Research Articles

High Correlation of Pre-Apheresis Peripheral Blood CD34+ Cell Counts with Final Apheresis Product CD34+ Cell Count by Using a More Accurate Determination of Patient Blood Volume

Abstract 2996The final apheresis product CD34+ cell count per Kg of recipient weight is used to determine if additional apheresis procedures are necessary to collect the targeted amount of hematopoietic stem cells for autologous peripheral blood stem cell transplantation. Flow cytometric analysis of CD34+ cells can take several hours. A more timely technique to predict apheresis product CD34+ cell counts during apheresis may help determine if further administration of cytokines is necessary or if apheresis catheters can be removed, and overall improve the efficiency of patient (pt) care. We performed a retrospective review of all pts undergoing autologous peripheral blood stem cell mobilization with granulocyte-colony stimulating factor (G-CSF) alone, G-CSF and plerixafor, or with chemotherapy followed by G-CSF, from July 2010 through May 2011 who underwent peripheral blood stem cell collection with apheresis on the COBE Spectra cell separator. Linear regression models were used to formulate the calculation of pt blood volume based on the pre-apheresis CD34+ cell count per micro liter of blood, the final apheresis product CD34+ cell counts, and the amount of blood processed during the apheresis procedure. This calculated blood volume is expressed by the formula BV = 82.5(patients weight in Kg) + 793. We then prospectively evaluated the next consecutive pts who underwent stem cell mobilization and apheresis in June and July 2011. Twenty-seven apheresis collections were done on 26 pts. Fourteen pts were female, and 12 pts were male. Seventeen pts were diagnosed with myeloma, 6 pts with NHL, and 3 pts with other diseases. Twenty pts were mobilized with G-CSF (10 ug/kg daily) with apheresis to begin on day 5. Fifteen of the 20 pts required plerixafor on day 4 because of low peripheral blood CD34+ cell counts (< 10/ul). Six pts were mobilized with chemotherapy followed by G-CSF 10 ug/kg daily until peripheral CD34 cell counts recovered greater than 10/ul and then apheresis was started. Each pt had their blood volume calculated according to the formula above and the peripheral blood CD 34+ cell count was measured on the first and second day of apheresis. The peripheral blood CD 34+ cell count/ul was multiplied by 1000 and this product was multiplied by the calculated blood volume and then divided by the pts weight [(PBCD34+ cell count × 1000) × BV]/Kg to determine the predicted apheresis product CD34+ cell count, which was then compared to the actual apheresis product final CD34+ cell count. On the first day of collection the mean for the predicted product CD34+ cell count was 4.98 × 106 +/− 3.1 × 106, and the actual apheresis product CD34+ cell count was 4.61 × 106 +/− 2.90 × 106 (Pearson correlation r value of 0.913 and a p value <0.001)(see figure). Nineteen collections were evaluable on the second day of collection with the mean for the predicted product CD34 + cell count of 2.08 × 106 +/− 1.64 × 106, and the mean for the actual apheresis product CD34+ cell count of 2.29 × 106 +/− 0.768 × 106 (Pearson correlation r value of 0.620 with a p value of 0.005). There was no significant difference in the correlation between patients mobilized with G-CSF alone, G-CSF and plerixafor or after chemotherapy and G-CSF. In conclusion, a more accurate determination of patient blood volume allowed for a high degree of correlation on the first day of peripheral blood stem cell collection on the COBE Spectra machine between the predicted product CD34+ cell count and the actual apheresis product CD34+ cell count. An accurate prediction of the final apheresis product CD34+ cell count may allow for less cytokine administration, quicker removal of apheresis catheters, and more efficient disposition of patients undergoing peripheral blood stem cell collection. [Display omitted] Disclosures:Shaughnessy:Otsuka: Honoraria, Speakers Bureau; Millenium: Honoraria, Speakers Bureau; Genzyme: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Prediction of Apheresis Peripheral Blood Stem Cell Yield Based on Pre Apheresis Absolute Peripheral Blood Stem Cell Counts,

Abstract 4047Stem Cell collection via apheresis is the preferred method of collecting stem cells for hematopoietic stem cell transplantation. Accurate prediction of stem cell yield is important for the planning of apheresis procedures and for the collection of adequate stem cells. Pierelli et. al. (Vox Sanguinis 2006, 91; 126) proposed a mathematical formula to estimate the CD34+ dose collected on 1st day of apheresis based on the peripheral blood (PB) CD34+ concentration pre-apheresis and the blood volume processed (BVP). Patients and Methods : We tested the predictive value of this formula in a multicenter sample of 1608 apheresis procedures performed at 5 centers (SCSP n=85, CAS n=195, HIAE n=305, China n=172, MDACC n=851). A 50% randomly selected sample of the MDACC study population was included in this analysis. Each contributing institution selected all consecutive apheresis procedures performed over a designated time frame. Baseline patient characteristics were collected including age, gender, diagnosis, mobilization regimen, complete blood counts on day of collection, and the absolute PB CD34+ counts within 24 hours of the first apheresis procedure. Subjects who did not have data on the PB CD34+ counts were not included in the analysis. Information on total BVP and CD34+ cells collected/kg was also collected. There were 694 males (59%), median age was 50 years. To facilitate comparison of results, we used the same statistical methods reported by Pierelli et. al. to assess the correlation between the actual (ay) and predicted (py) CD34+ yields, including assessment of the linear correlation between these measures and the distribution of the ay/py ratio. Results: Data on both actual and predicted yields were available for 1148 (97%) records. Overall, Pearson’s correlation coefficient (r) between ay and py was 0.67, ranging from 0.67 for MDACC to 0.86 for China and CAS. Median ay/py ratio was 1.1 (0.02–433) ranging from 0.99 for HIAE to 1.5 for China and CAS. To characterize the correlation between ay and py and facilitate the clinical application of our findings, we classified the actual and predicted yields as falling below (<2), within (2–5), or above (>5) the conventionally acceptable collected CD34+ doses (x106/Kg). Positive predictive value (PPV) of py was estimated considering the distribution of ay as the “gold standard”. PPV was relatively high for py >5 [85% (95% CI 81–89%)], average for py <2 [72% (95% CI 68–76%)], and low for py between 2 and 5 [56% (95% CI 51–62%)]. This pattern was consistent across institutions. PPV ranged from 55% (SCSP) to 80% (China) for py<2; from 37% (CAS) to 68% (MDACC) for py between 2 and 5, and from 73% to 94% for py>5, exceeding 80% at all institutions except SCSP. Overall, 13% of cases predicted to have a CD34+ yield between 2 and 5, had an actual yield <2. This proportion varied across institutions ranging from 5% at SCSP to 28% at HIAE. Notably, the distribution of BVP (ml/Kg) was comparable across the 3 categories of ay defined above with a median of 204 (range 74–263). Consistent pattern was also observed within institutions. Conclusion: Our data indicate that the formula of Pierelli et.al. is associated with high PPV for predicted CD34+ doses >5, acceptable PPV for doses<2, and relatively low PPV for doses falling between 2 and 5. The data also suggests that CD34+ yields correlate with pre-apheresis CD34+ count and are independent of BVPNTotal 1182rangeSCSP 85rangeCAS 195rangeChina 172rangeHIAE 305rangeMDACC 425rangeAbsolute PB CD34+ × 106/μl (median, range)280.3–2735314.2–279251.3–1750140.3–411310.6–604312–2735Weight, Kg (median, range)729–1696738–143619–1106322–1007313–1438411–169Day 1 Blood volume processed, L (median, range)153–131148–25123–299.54.5–12.5195–131168–27Actual Day 1 CD34+ dose (x 106/Kg), (median, range)2.90.01–1092.90.2–293.80.01–1091.80.01–503.50.1–442.80.2–101<236%27%33%53%34%33%2–531%40%28%20%27%39%>533%33%38%27%39%28%Predicted Day 1 CD34+ dose (× 106/Kg), (median, range)2.40.02–2112.60.3–242.20.1–730.960.02–253.20.1–672.40.1–211<244%45%44%63%33%42%2–530%25%37%17%28%35%>527%30%19%20%38%23%Ratio CD34+ dose collected/predicted1.10.02–4331.10.2–111.50.03–6.31.50.02–4330.990.04–121.10.1–9.9Pearson's correlation coefficient0.670.830.860.860.770.67Pearson's within Predicted Day 1 dose<20.420.080.630.50.570.282–50.280.250.280.20.360.25>50.560.770.810.70.630.6 Disclosures:No relevant conflicts of interest to declare.

Effectiveness and Potential Strategies to Improve Outcomes Following Chemomobilization with Etoposide + G-CSF in Patients Undergoing Autologous Stem Cell Transplantation for Lymphoma

Abstract 2083 INTRODUCTION:The addition of chemotherapy to G-CSF for stem cell mobilization prior to autologous stem cell transplantation (ASCT) provides the potential for increased cell yield and improved mobilization outcomes relative to G-CSF alone. We have investigated the use of mid-dose VP-16 plus G-CSF in pts with lymphoma and examined whether plerixafor might be incorporated into this chemomobilization backbone in a cost-effective way for a population with inferior outcomes. METHODS: Between June 2004 and September 2010, 159 pts with lymphoma underwent ASCT following the use of VP-16 (375mg/m2 on D#1 and D#2) and G-CSF (5mcg/kg twice daily from D#3 through the final day of collection) for mobilization. 26 pts also received a dose of Rituximab (375mg/m2) on D#1. Stem cell collection was initiated when the peripheral blood CD34 cell count was more than 7 per ul. Data on costs for fixed and variable expenditures associated with mobilization and collection were calculated on an individual patient basis. Costs also included unexpected complications such as inpatient hospitalizations, antibiotic use and blood product transfusions. “Poor mobilizers” were defined as pts failing to collect 5 × 106 cells in one or two days. Univariable and multivariate logistic regression were performed to identify predictive models for poor mobilization and to identify hypothetical breakpoint scenarios for the cost-effective utilization of plerixafor. For the breakpoint scenarios, a median of 3 doses of plerixafor was assumed based on the published phase III data with plerixafor plus G-CSF. RESULTS: Of 159 pts with lymphoma, 90 (57%) were identified as “good mobilizers,” 43% were “poor mobilizers”, and 150 (94%) collected at least 2 × 10 6th/kg CD34 cells in total (83% within 4 apheresis sessions), comparing favorably to published data with G-CSF alone or G-CSF + plerixafor. 51 (32%) required PRBC or platelet transfusion, 10 (6%) were admitted to the hospital during the mobilization period, and 8(5%) required a second mobilization or bone marrow harvest. There was no increased incidence of secondary malignancies. Average costs were $14923 ($6121-$24546) for good mobilizers and $27044 ($12206-$51846) for poor mobilizers (p<0.05). The first peripheral blood CD34 count (obtained between D9-D15, with 82% of first counts obtained on D12), accurately predicted “good” vs “poor” mobilizers (c statistic 0.941, CD34 cutpoint 27/uL). Using our data, we estimated that it would not be cost effective to give plerixafor to all patients, even if 100% of patients subsequently became “good” mobilizers (net loss $15,817/pt). Instead, by reserving plerixafor for only predicted “poor” mobilizers (probability<0.5) at the time of first CD34 count, we estimated that 64% (n=49) of predicted “poor” mobilizers would need to become “good” mobilizers in order to achieve cost neutrality. CONCLUSION: VP-16 and G-CSF is a safe and effective mobilization regimen for pts with lymphoma and compares favorably to published data with G-CSF alone or G-CSF + plerixafor. Mobilization outcomes after chemomobilization might be further improved in a cost-effective way by adding plerixafor in patients predicted by the first peripheral blood CD34 count to be poor mobilizers. This will be investigated prospectively. Disclosures:Shea:Otsuka Pharmaceuticals: Research Funding.

Rapid Disease Progression in HIV-1-Infected Men Who Have Sex with Men Is Negatively Correlated with Peripheral Plasmacytoid Dendritic Cell Counts at the Early Stage of Primary Infection

To explore the relationship between absolute dendritic cell (DC) counts at the early stage of primary human immunodeficiency virus type 1 (HIV-1) infection (PHI) and subsequent disease progression, we performed a prospective study of 16 rapid progressors (RPs) and 12 typical progressors (TPs) from a PHI cohort of men who have sex with men. Significantly decreased plasmacytoid DC (pDC) and myeloid DC (mDC) counts in the blood of RPs were observed at study entry as compared with TPs and healthy HIV-1-negative subjects. Low baseline pDC counts were significantly associated with rapid disease progression after adjustment for baseline CD4(+) T cell counts, mDC counts, and HIV-1 load. Kaplan-Meier survival analysis showed that low pDC counts were strongly associated with rapid disease progression. Our findings demonstrated the predictive value of blood absolute pDC counts at baseline in PHI for HIV-1 disease progression. Further studies are required to confirm this notion.

High detection rate of Trichomonas vaginalis in benign hyperplastic prostatic tissue

While Trichomonas vaginalis, a protozoan parasite, is a well-investigated pathogen in the female population, there is little awareness of its significance in the male uro-genital tract. The presence of T. vaginalis in the prostate gland has only been scarcely investigated and has never been attested in conditions other than clinical prostatitis. Still, by some authors, this organ is regarded as ecologic niche for T. vaginalis. Since normal prostate tissue of sufficient quality is hard to come by, we investigated samples from 86 patients (mean age 68.7±7.6years) suffering from benign prostatic hyperplasia (BPH), a medical condition currently ranked as noninfectious, but characterized by chronic inflammatory tissue infiltrates of unknown etiology. Applying two different PCR protocols and sequence analysis of the respective amplicons, we detected T. vaginalis DNA in 29/86 (34%) BPH tissue samples, whereas in only 2/86 (2.3%) cases T. vaginalis grew in culture. Detection of T. vaginalis DNA correlated significantly (P<0.01) with elevated peripheral blood monocytic cell counts, appearing along with protozoan infections. Given the unexpected high prevalence of T. vaginalis in BPH tissue of a nonselected, elderly study population from Austria, further epidemiological studies have to confirm this finding. Potential interactions of T. vaginalis in its prostatic habitat may be investigated with respect to their possible contribution to the inflammatory pathogenesis of BPH, since inflammatory cytokines have been shown to sustain prostatic hyperplastic growth.

Peripheral Blood Stem Cell Collection In Patients Undergoing Induction Therapy with Lenalidomide Based Regimens: Failure Rates and Salvage Approaches

AbstractAbstract 2253 Background:Lenalidomide based combinations are among the most common initial therapies for myeloma. Previous studies have suggested that lenalidomide therapy can result in suboptimal stem cell collection in patients eligible to undergo autologous stem cell transplantation, especially older patients after prolonged exposure to the drug. Many salvage approaches are used when attempting repeat stem cell collection in this patient group. Patients and Methods:Two hundred twenty four patients who underwent stem cell collection following lenalidomide-dexamethasone induction from July 2004 and December 2009 were included in the current analysis. Data pertaining to the duration of lenalidomide therapy, stem cell mobilization regimen, and the collection yields were collected from the medical records. Results:The median age at mobilization was 60.6 years (range; 29, 76) and 136 (60%) were male. There were a total of 245 collection attempts from among 224 patients, 21 (9.8%) patients attempting to remobilize after failing to collect the desired numbers of stem cells at the first attempt. We first analyzed the results of the initial collection attempt. The median duration of lenalidomide therapy prior to stem cell collection was 4 months (range; 1, 26). The mobilization strategies were GCSF (Granulocyte Colony Stimulating Factor) alone in 151 (67%) patients, cyclophosphamide (CTX) followed by GCSF in 29 (13%) patients, and GCSF plus AMD3100 in 44 (20%) patients. Among those receiving AMD3100, it was added either due to peripheral blood CD34 cell count not reaching the threshold for initiation of harvest or for poor first day CD34 cells collection in 34 patients and given in a planned fashion in 10 patients. Overall 15 patients (7%) failed to reach the peripheral CD34 cell counts required to initiate apheresis, and among those starting apheresis 6 patients failed to collect at least 2 million CD34 cells/kg; a cumulative failure rate of 9%. Another 18 (8%) patients failed to collect at least 4 million CD34 cells /kg. The CD34 cells yield on day 1, the total yield, number of collections, the average daily yield and the percentage of the targeted cells collected for each mobilization strategy including failure rates are detailed in the table.Twenty-one patients reattempted stem cell mobilization; including 14 that failed a first attempt and 7 did who not achieve the intended goal even though they collected more than 2 million CD34 cells/kg. The mobilization regimens were GCSF alone, CTX + GCSF, GCSF + GM-CSF (Granulocyte Macrophage Colony Stimulating Factor) and GCSF + AMD in 5, 8, 3, and 4 patients respectively. All patients collected at least 2 million CD34 cells /kg and 14 patients (70%) collected more than 4 million CD34 cells /kg. The median CD34 cells collected with the second attempt was 5.4 million/kg (rang; 2, 19.5) bringing the median total collection for these 21 patients to 9.6 million/kg (2.6-19.6). Overall, of the 224 patients studied, all but the 6 patients who failed initially and did not attempt a second collection collected at least 2 million CD34 cells /kg and 197 (88%) collected at least 4 million CD34 cells/kg. Conclusion:While the overall failure rate of stem cell collection in patients receiving initial therapy with lenalidomide is 10%, a risk adapted approach of adding AMD3100 appear to decrease the risk of failure. However, majority of patients failing a stem cell harvest attempt can be salvaged with a second collection allowing these patients to proceed to a stem cell transplant if desired.AllG-CSFCTX +G-CSFAMD upfrontAMD rescuePN224151291034Day 1 CD34 collection Median(Range)2.0 (0–28.6)2.1 (0–10.1)4.2 (0–28.6)1.9 (0.7–15.3)1.3 (0.09–8.6)<0.001Total CD34 collection Median(Range)7.6 (0–28.6)7.5 (0–15.6)9.3 (0–28.6)8.5 (4.2–15.3)6.5 (0.2–13.5)0.02Number of collection days Median(Range)4 (0–12)4 (0–12)2 (1–11)2.5 (1–7)4 (1–9)<0.001Average daily collection Median(Range)1.6 (0–28.6)1.5 (0–10.1)4.4 (0–28.6)2.3 (1.6–15.3)1.2 (0.1–8.6)<0.001% of goal collected Median(Range)100 (0–715)97 (0–233)114 (0–715)113 (82–172)106 (2.5–141)0.001% patients failed (no apheresis or CD34 <2 million/kg)9111006NS% patients <4 million/kg172014015NS Disclosures:Gertz: Celgene: Honoraria; Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genzyme: Research Funding. Lacy: Celgene: Research Funding. Dispenzieri: Celgene: Honoraria, Research Funding; Binding Site: Honoraria. Micallef: Genzyme: Membership on an entity's Board of Directors or advisory committees. Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Cholinergic activation of hematopoietic stem cells: role in tobacco-related disease?

Tobacco use is associated with an increase in the white blood cell (WBC) count. This association has been attributed to bronchopulmonary inflammation and/or infection. It is not known if nicotine itself may play a role. The objective of this study was to determine whether nicotine itself could affect the WBC count, and to determine whether this was due to a direct effect on hematopoietic stem cells (HSC). C57Bl6J mice received nicotine orally, and measurements of the WBC count, bone marrow and spleen cellularity, and HSC count were made. To determine the functionality of HSCs, irradiated animals received bone marrow transplants from vehicle or nicotine-treated mice. Nicotine increased leukocytes in the peripheral blood, bone marrow and spleen. The peripheral red cell and platelet count were unaffected. Nicotine increased the frequency of HSC in the bone marrow. Isolated long-term HSCs from nicotine-treated mice transplanted into irradiated mice regenerated all hematopoietic cell lineages, demonstrating the functional competence of those HSCs. HSCs expressed nicotinic acetylcholine receptors (nAChRs), as documented by FITC-conjugated alpha-bungarotoxin binding. Nicotine increased soluble Kit ligand, consistent with stem cell activation. In conclusion, the data suggest a new mechanism for the increased WBC associated with tobacco use. The effect of nicotine to activate hematopoiesis may contribute to tobacco-related diseases.

Intrinsic and extrinsic effects of mafG deficiency on hematopoietic recovery following bone marrow transplant

MafG is the small subunit of the transcription factor NF-E2 that controls terminal megakaryocyte maturation and platelet release. Studies were conducted to evaluate the intrinsic and extrinsic effects of mafG deficiency on bone marrow engraftment kinetics. We used mafG knockout mice either as donors or recipients in bone marrow transplantations with wild-type mice and compared the engraftment kinetics to transplantations using wild-type donors and recipients. We measured peripheral cell counts, the presence of circulating donor-derived cells by flow cytometry, changes in the cellularity of the bone marrow and splenic weight on day 5, 7, 14, and 1 month post-transplantation. Compared to wild-type recipients, mafG recipients had delayed platelet and leukocyte recovery and lower spleen weight at early time points after transplantation. Intrinsic effects: When mafG-deficient bone marrow served as donor source, we observed more rapid recovery of bone marrow cellularity and increased splenic hematopoiesis. The finding of increased short-term hematopoietic stem cells and progenitors in the mafG-deficient bone marrow could explain the accelerated hematopoietic recovery after transplantation. Furthermore, the expression of Bach 2, which can form a heterodimer with mafG protein, was found to be greatly reduced, while Notch 1 expression was increased in mafG-deficient mice. Extrinsic effects: When mafG-deficient mice were transplant recipients, there were delays in recovery of normal levels of marrow and splenic hematopoiesis as well as circulating leukocytes and platelets. Our study demonstrates that mafG expression has intrinsic and extrinsic effects on hematopoietic engraftment following bone marrow transplantation.

Despite being highly diverse, immunovirological status strongly correlates with clinical symptoms during primary HIV-1 infection: a cross-sectional study based on 674 patients enrolled in the ANRS CO 06 PRIMO cohort

To analyse immunovirological status during primary HIV-1 infection (PHI) according to contemporary clinical status and time since infection. Plasma HIV-RNA and peripheral blood mononuclear cell (PBMC) HIV-DNA levels and CD4 cell counts were determined at enrolment in the ANRS PRIMO cohort. Time since infection was estimated based on both the number of antibodies on western blot at enrolment (0-1, 2-4 or > or =5 specific antibodies) and the estimated interval between infection and enrolment based on clinical and epidemiological features. Patients were classified according to the presence or absence of clinical symptoms at enrolment. Between 1996 and 2006, 674 patients were enrolled an estimated median of 47 days after infection. Median marker values were as follows: HIV-RNA 5.10 log(10) copies/mL (range <1.70-8.33); HIV-DNA 3.30 log(10) copies/10(6) PBMCs (<1.84-4.93); and 506 CD4 cells/mm(3) (40-1542). Median HIV-RNA and PBMC HIV-DNA levels were significantly higher in patients with 0 or 1 specific antibody (n = 71) than in patients with 2-4 (n = 228) or > or =5 antibodies (n = 375). Symptomatic patients had significantly higher HIV-RNA and PBMC HIV-DNA levels and lower CD4 cell counts. However, 10% of symptomatic patients recruited shortly after infection had favourable immunovirological status. Plasma HIV-RNA, PBMC HIV-DNA and CD4 cell count values were highly diverse and correlated strongly with clinical status during PHI. Early diagnosis was not always associated with severe PHI. Combining PBMC HIV-DNA with HIV-RNA, CD4 cell count and clinical symptoms would have allowed identification of 179 patients (26.5%) at high risk of rapid disease progression who did not meet current guidelines for early treatment initiation.

Bone marrow biopsy in patients with hepatitis C virus infection: Spectrum of findings and diagnostic utility

Patients with hepatitis C virus (HCV) infection develop a number of hematologic disorders, with benign and malignant B-cell proliferations being the most common. HCV-infected patients are also prone to developing peripheral cytopenias, the etiologies of which are multifactorial and include hypersplenism and/or antiviral medications. Some of these patients may undergo bone marrow biopsy but no study has systematically recorded the bone marrow findings in this patient group. Here, we report on the range of bone marrow findings in 47 adult HCV-infected patients. These patients, who lacked concurrent human immunodefiency virus (HIV) infection, most commonly presented for a bone marrow biopsy due to abnormal peripheral cell counts. The bone marrow biopsies displayed a range of findings. Dyserythropoiesis, present in 19% of the cases, was the most common finding. Patients with pancytopenia(n = 6), as defined by current World Health Organization standards, were the most likely to have bone marrow abnormalities; two pancytopenic patients had acute myeloid leukemia, and one patient had a primary myelodysplastic syndrome. There was no correlation in bone marrow findings and antiviral medications, MELD score, cirrhosis or splenomegaly, suggesting that the degree of bone marrow dysfunction is independent of stage of HCV. The results of this study suggest that bone marrow biopsy in HCV-infected patients, even those with features of hypersplenism and/or documented antiviral therapy, can be a valid test for hematologic evaluation, especially for patients with severe pancytopenia and/or sudden alterations in peripheral cell counts.

Road Transportation Stress Promptly Increases Bovine Peripheral Blood Absolute NK Cell Counts and Cortisol Levels

Livestock transportation effects on the number of circulating leukocytes have been reported. However, data related specifically to the relation between acute stress levels during transport and leukocyte differentiation, including lymphocyte subsets, are lacking. This study was undertaken to evaluate the distribution of peripheral blood leukocyte differential counts, CD25+ lymphocytes and NK cells in calves subjected to truck transportation on different road types. Healthy Japanese Black calves were divided into three treatments: 1) those moved around in a mountainous area (Group M); 2) those moved around on flatland (Group F); and 3) those that were not transported (control). The plasma cortisol levels in Group M increased during transport. The increase was significantly higher at the end of transport than in the controls (P<0.05); a slight increase was noted in Group F. Total leukocytes and the neutrophil to lymphocyte ratio in Group M were elevated with neutrophilia at 2 hr post-transport (P<0.05); the former levels remained higher than those in the controls for 4 hr. The numbers of lymphocytes, monocytes, eosinophils and CD25+ lymphocytes remained unchanged throughout the observations. The number of circulating NK cells in Group M increased during transport and peaked shortly after transport (P<0.05). Subsequent to these time points, the counts in Group F showed a trend toward elevation. The circulating NK cell counts were positively correlated with the plasma cortisol level during transport (M, r=0.755, P<0.0005; F, r=0.653; P<0.005). These results suggest that circulating NK cells might be more rapidly mobilized than other leukocytes. Therefore, they might reflect acute stress levels in calves during road transportation.

Targeting Trojan Horse leukocytes for HIV prevention

…. is sure design'd, by fraud or force: trust not their presents, nor admit the horse. Virgil, Aeneid Human immunodeficiency virus type 1 (HIV-1), the lymphotropic virus that causes AIDS, has infected more than 60 million people worldwide since its clinical appearance in 1981. Despite intensive prevention efforts, the HIV/AIDS epidemic continues to spread, particularly in developing countries in sub-Saharan Africa, southeast Asia and the Caribbean, as well as the developed world [1]. Although HIV can be transmitted very efficiently parenterally, the advent of routine blood screening prior to transfusion and harm reduction programs for injection drug users, have made this mode of transmission much less common than mucosal transmission. Most new HIV infections are attributable to mucosal transmission: through genital and rectal mucosae in the case of sexual transmission and through oral or gastrointestinal mucosae in the case of mother-to-child transmission [2]. Much has been learned about HIV pathogenesis and infection mechanisms at the molecular level, but the scientific community has yet to develop an effective vaccine or microbicide for HIV prevention. Many unanswered questions remain concerning HIV-1 sexual transmission. In 1983, barely 2 years into the AIDS epidemic, we hypothesized that the agent that was subsequently identified as HIV-1 may be sexually transmitted by infected ‘Trojan Horse’ leukocytes in semen [3]. This hypothesis was based on our knowledge at the time that human semen contains substantial numbers of T lymphocytes and macrophages, which could host a T-cell tropic virus, and the following assumptions: intracellular virus would be better protected than free virus from adverse effects of antiviral factors in the genital environment such as antiviral antibodies likely to be present in genital secretions of the virus-infected transmitter, as well as antimicrobial peptides that play an important role in genital innate immune defense; and virus-infected allogeneic cells could also escape early detection by major histocompatibility complex (MHC)-restricted cytotoxic T cells in a new host. Over the intervening 25+ years, others have also championed this cause [4,5], and convincing evidence has emerged from clinical research as well as in-vitro and animal studies that infected leukocytes indeed play a role in HIV transmission. Yet, most recent research on sexual HIV transmission has focused on cell-free HIV in genital secretions because of the wide availability of HIV RNA quantification assays. Furthermore, the majority of HIV vaccines and microbicides have been designed to block transmission of cell-free virus and have been tested in animal and in-vitro models that use cell-free virus as the only infectious inoculum. As the molecular events underlying cell-associated HIV transmission differ from those underlying cell-free virus transmission, many of the current vaccine and microbicide candidates might not be expected to protect against cell-associated HIV transmission. The failure of several recent vaccine and microbicide clinical trials may be due in part to this oversight. It should be possible to design strategies that block cell-associated HIV transmission as well as cell-free HIV transmission. In this article, we present an overview of research that has been conducted on cell-associated HIV mucosal transmission and recommendations for future research. We focus on sexual HIV transmission, but this review also has relevance for mother-to-child HIV transmission, which may occur through mucosal transmission of cell-associated HIV from maternal genital or mammary gland secretions [6–8]. We review published reports that describe and enumerate HIV-infected cells in genital secretions, and compelling evidence from clinical, animal and in-vitro studies demonstrating that such cells can transmit HIV across genital tract epithelial surfaces; potential molecular mechanisms underlying cell-associated HIV transmission that could be specifically targeted by future HIV prevention strategies; and in-vitro and animal cell-associated HIV transmission models currently used for studies on cell-associated HIV transmission mechanisms and for testing vaccine and microbicide candidates. Using this information as a foundation, we discuss the evidence and probability that various current microbicide and vaccine approaches prevent cell-associated HIV transmission, and suggest additional microbicide and vaccine concepts and experiments that will move this field forward.

Frequency and prognostic significance of murine double minute protein-2 overexpression and p53 gene mutations in childhood acute lymphoblastic leukemia

Aim: Determination of frequency and prognostic significance of murine double minute protein-2 (MDM-2) over expression and its association with p53 status in children with acute lymphoblastic leukemia (ALL).Methods: MDM-2 expression by flow cytometry and p53 gene status by PCR were determined in peripheral blood or bone marrow of 46 ALL children (at initial diagnosis) and control group.Results: MDM-2 was significantly overexpressed in 15 patients (32·6%). p53 mutation was detected in six out of 46 patients at initial diagnosis, three of them were out of 29 cases achieving complete remission (CR) and the other three cases were out of 17 of relapsed patients, which is significantly higher than CR group (P<0·05). Positive correlation was found between the MDM-2 overexpression and initial WBCs count, peripheral blast cell count and presence of CNS blasts (P<0·05, <0·05 and <0·05 respectively).Conclusion: MDM-2 is overexpressed in a significant number of childhood ALL, and more often observed in the poor outcome group and its frequency is not related to p53 status. Measurement of MDM-2 as a bad prognostic marker even in cases with non-mutant p53 is very important. Moreover, MDM-2 may be a potential molecular target for production of new cancer therapy.

A Case of Massive Intracerebral Haemorrage in Advanced Myelofibrosis Due to Atypical Metaplasia.

Abstract 4988 BACKGROUNDPrimary myelofibrosis is a chronic myeloproliferative disease characterized by connective tissue deposition in the bone marrow and extramedullary hematopoiesis commonly involving the liver and the spleen. CASE REPORTWe report the case of a 60 year old patient with advanced Post Thrombocitemia Myelofibrosis extensively localized in almost all organs. He was referred to our Center in April 2008; clinical, radiological and haematological features suggested an evolution into Myelofibrosis. Molecular analysis for V617F JAK2 mutation was negative. Hemogram showed leukocytosis, mild anemia and normal platelet count. After few months of treatment with Danazol and Thioguanine, he was admitted for hemodynamic failure; echocardiography and chest X-ray were normal; abdomen CT scan confirmed important splenomegaly and didn't show any deep thrombosis. During hospitalization he developed massive intracerebral haemorrhage, cause of death. Post mortem histopatological examination documented osteomyelosclerosis and wide localization of extramedullary hematopoiesis. Morphological examination of the specimens showed an extensive dissemination of megakaryocytes, erythroid and myeloid precursors immunoreactive with factorVIII, hemoglobin, and myeloperoxidase respectively. We documented intravascular involvement of spleen, liver, lymph nodes, lungs, adrenal glands, myocardium, and central nervous system. Noteworthy fibrosis associated with haematopoietic cells were broadly distributed into retroperitoneal tissue. CONCLUSIONSDespite the massive myeloid metaplasia shown by the autopsy, general conditions of the patient until hospitalisation were good. Physical and radiological examination didn't reveal vascular and organ involvement. Although myeloid metaplasia is a possibility in advanced myelofibrosis, the assessment of cerebral and myocardial localization is difficult to find and standard radiological exams are not useful. In the subset of patients with Myelofibrosis and unexplained organ failure it could be interesting to look for an intravascular metaplasia throughout a multi-step approach: i) CD34+ peripheral cells count, since a very high count of these cells could suggest an extensive metaplasia; ii) high resolution pulmonary CT scan; iii) perfusional myocardial and/or pulmonary scintigraphy. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Efficacy and Safety of VP-16 and G-CSF as a Mobilization Regimen Prior to Autologous Stem Cell Transplantation (ASCT) for Patients (Pts) with Multiple Myeloma (MM) and Lymphoma.

Abstract 4225 INTRODUCTIONAlthough the number of ASCTs performed for myeloma and lymphoma continues to increase, there remains little consensus about the best means for mobilizing stem cells prior to transplantation and whether cytokine alone or cytokine plus chemotherapy is preferred. Cytokine alone, usually G-CSF, provides easy scheduling while the combination approaches generally provide higher cell yields and additional cytoreduction. Because of its value as a cytoreductive agent, and its lack of stem cell damaging properties or other toxicities such as hemorrhagic cystitis or significant liver toxicity, we investigated the use of mid-dose VP-16 plus G-CSF as a mobilizing regimen. Here, we report our experience with this regimen prior to ASCT for pts with various diseases. METHODSBetween May 2004 and June 2009, 296 pts with the following diseases underwent autologous stem cell transplantation following the use of VP-16 (375mg/m2 on D#1 and D#2) and G-CSF (5mcg/kg twice daily from D#3 through the final day of collection) for mobilization: Multiple Myeloma (152), Non-Hodgkin Lymphoma (98), Hodgkin Disease (38), Acute Promyeloyctic Leukemia (4), Acute Myelogenous Leukemia (1), Chronic Lymphocytic Leukemia (1), Plasma Cell Leukemia (1), and testicular cancer (1). In 14 pts, one dose of Rituximab (375mg/m2) was also given on D#1. 125 pts were female, 171 were male, and median age at the time of transplant was 55 years (range 19-79). Stem cell collection was initiated when the peripheral blood CD34 cell count was more than 7 per microliter. The target total CD34 cell collection was 5 × 106 cells. RESULTSAmong all pts, 286 (97%) had stem cells successfully collected after one mobilization. An additional 7 (2%) pts required more than one mobilization or bone marrow harvest, with one pt experiencing progressive disease after failed mobilization. 117 (40%) pts required either treatment for infection (inpatient hospitalization and/or intravenous antibiotics; 14% of all pts) or at least one supportive transfusion with PRBCs or platelets (33% of all pts). 1 pt is known to have developed secondary myelodysplastic syndrome, and there are no known cases of secondary AML. The median number of days collecting stem cells was 1 for MM pts and 2 for lymphoma pts, and the median total number of CD34 cells collected was 12 × 106 for MM pts and 6 × 106 for lymphoma pts. 94% of MM pts and 43% of lymphoma pts collected in a single day. The median time to neutrophil engraftment was 11 days for both MM and lymphoma pts, and the median time to a sustained platelet count > 20,000 without transfusions was 16 days for MM pts and 15 days for lymphoma pts. “Poor mobilizers” were defined as pts who failed to collect 5 × 106 cells in one or two days, and included 19% of all pts. 1% of MM pts and 40% of lymphoma pts were “poor mobilizers,” a statistically significant difference (p<0.001). 6 lymphoma pts failed to collect at least 2 × 106 cells, though all MM pts achieved this minimum target. The pre-mobilization white blood count (p=0.01) and platelet count (p=0.006) were associated with poor mobilization in lymphoma pts by univariable logistic regression analysis. Pts requiring more than 14 days to engraft neutrophils or 27 days to engraft platelets (> 1 S.D. above the mean) were considered “poor engrafters,” and included 16 pts (6%). We failed to detect statistically significant associations between “poor engrafters” (n=6 for lymphoma, n=10 for MM) and the covariates of “poor mobilizers,” age, disease type, amount and type of prior chemotherapy, radiation therapy, marrow cellularity or disease involvement, or the pre-mobilization complete blood count. CONCLUSIONVP-16 and G-CSF is an effective and safe mobilization regimen for pts undergoing ASCT with a low incidence of requiring hospitalization and/or IV antibiotics for fever, or of association with subsequent myelodysplasia or secondary AML. A diagnosis of lymphoma (HD or NHL) as opposed to MM, and low platelet or WBC count pre-mobilization predicted for poor CD34 cell yield. Further research is needed to help predict “poor mobilizers” and “poor engrafters” prior to transplant, as are the use of novel agents, such as the CXCR4 antagonist plerixafor, to improve outcomes in these populations. Disclosures:Shea:Genzyme, Millenium, Otsuka: Research Funding; Board of Directors, CALGB; CIBMTR Scientific Advisors, President-elect: Membership on an entity's Board of Directors or advisory committees; Genzyme, Genentech, Novartis: Consultancy.

Appropriate Timing of G-CSF Use After Mobilization Chemotherapy Significantly Increases the Yield ofCD34+ Cells in autoPBSCT.

Abstract 2144Poster Board II-121The yield of CD34+ cells collected by apheresis for autologous peripheral blood stem cell (PBSC) transplantation was greatly increased when the appropriate timing was determined to begin using G-CSF after COAEP mobilization. 29 patients with lymphoma or multiple myeloma (MM) received the same mobilization chemotherapy, including CTX 400mg/m2 d1; VDS 2 mg/ m2 d1; Ara-C 60 mg/m2 ×5d; vp-16 60 mg/m2 ×5d; and prednisone 40 mg/m2 ×5d. The control group (12 cases) received subcutaneous G-CSF (filgrastim) at the first restoration after the initial nadir of the peripheral WBC count. The experimental group (17 cases) received G-CSF during the steady rise of the WBC count (end of fluctuating after initial nadir). G-CSF was given in a single daily subcutaneous dose of 300μg until the final PBSC apheresis.When the peripheral WBC and mononuclear cell (MNC) counts reached 10.0×109/Land 1.0×109/L, respectively, leukapheresis was carried out using the COBE Spectrablood cell separator. Despite comparable treatment with alkylating agents, a significantly increased yield of CD34 positive cells was observed in the experimental group (32.0×106/kg) compared to the control group (3.1×106/kg) (P=0.0182). This result indicates the importance of appropriate timing for the use G-CSF after mobilization chemotherapy to increase the CD34+ cell yield. Disclosures:No relevant conflicts of interest to declare.

Discontinuation of Imatinib Therapy After Achieving a Molecular Response in Chronic Myeloid Leukemia Patients.

Abstract 859▪▪This icon denotes an abstract that is clinically relevant. BackgroundImatinib (IM) has greatly improved survival rates in chronic myeloid leukemia* (*CML). However, all patients (pts) must continue treatment for an unknown period of time.A pilot study of the first pts who discontinued IM therapy was previously reported (Rousselot et al. Blood 2007;109:58–60). The multicentre study Stop Imatinib (STIM) was initiated in July 2007 in order to evaluate the persistence of complete molecular remission (CMR) after stopping IM, and to determine the factors that could be associated with CMR persistence. MethodsInclusion criteria were IM treatment duration of at least 3 years and sustained CMR. Sustained CMR was defined as BCR-ABL/ABL levels below a detection threshold corresponding to a 5-log reduction (undetectable signal using RQ-PCR) for at least 2 years. Molecular relapse, defined as RQ-PCR positivity, was taken into account if confirmed in two successive assessments. In cases of molecular relapse, pts were re-challenged with IM at 400 mg daily. ResultsFrom the pilot study, 8 among 15 patients are still in CMR with a median follow up of 42 months (range 37-49). The number of patients enrolled in the STIM study was 69. 34 patients had received interferon alpha (IFNa) prior to IM and 35 pts were de novo. Median follow-up (range) was 17 months (6-24). 37 pts relapsed (loss of CMR) within the first 6 months and two patient relapsed after more than 6 months (M7 ,M18). At M12, the probability of remaining in CMR was 45% (95% CI: 33-56%). For previously treated with IFN (n=34) this probability was 44% (95% CI: 27-59%) versus 46% (95% CI: 29-61%) for de novo pts (p=0.93, overall). All patients in molecular relapse were sensitive again after imatinib re-challenge (decreasing BCR-ABL level, achievement CMR again). Male pts had a better probability of survival without molecular relapse (p=0.02) and a trend was observed for the low Sokal risk group (p = 0.06). Peripheral NK cells counts prior to IM discontinuation were significantly lower in relapse pts (mainly cytotoxic cells CD56dim) as compared to the non relapse pts(p=0.005). ConclusionsWe have confirmed that CMR can be sustained after discontinuation of imatinib with a long follow-up, particularly in male patients and in pts with cytotoxic NK cells in their peripheral blood. Using stringent criteria, it is possible to stop treatment in patients with sustained CMR, even in those treated with IM as a single agent.(ClinicalTrials.gov number, NCT00478985 [ClinicalTrials.gov]) Disclosures:Mahon:Amgen: Honoraria; Novartis Pharma: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria. Guilhot:Novartis Pharma: Consultancy, Honoraria; BMS: Consultancy, Honoraria.

Effects of leptin Arg25Cys on peripheral mononuclear cell counts and antibody response to vaccination in beef cattle

A single nucleotide polymorphism (SNP) in the leptin gene that results in Arg25Cys has been associated with beef carcass quality and milk composition in dairy cattle. However, leptin (LEP) also plays a role in immune performance and hence it was important to determine whether selection based on this SNP would negatively affect immune cell numbers or antibody production. LEP c.73C>T was assessed for effects on immune cell counts and antibody titres in 27 beef cattle herds (n = 556). A commercial rabies vaccine had been administered to these animals. Prior to being vaccinated, counts of several important mononuclear cells (total and activated B lymphocytes, total and activated T helper and T cytotoxic, WC1 T lymphocytes and monocytes) as well as baseline serum antibody titres were determined for each animal. On day 21, antibody titres were measured and a booster vaccine was administered. Finally on day 42, antibody titres and mononuclear cell types were again counted. Counts of six different cell types were significantly associated with the LEP genotype; however, no consistent patterns were observed between LEP genotype (TT, CT or CC) and peripheral blood mononuclear cell populations. Significant differences in the production of rabies antibodies in response to vaccination were observed relative to LEP genotype. Our results suggest that selection for either the C or T allele would not detrimentally impact on the measured indicators of immune function in beef cattle.

High-Risk Childhood Acute Lymphoblastic Leukemia

Although most children with acute lymphoblastic leukemia (ALL) are cured, certain subsets have a high risk of relapse. Relapse risk can be predicted by early response to therapy, clinical and pharmacogenetic features of the host, and genetic characteristics of leukemic cells. Though early treatment response can be assessed by the peripheral blast cell count after 1 week of single-agent glucocorticoid treatment or percent of bone marrow blasts by morphology after 1 or 2 weeks of multiagent induction treatment, determination of minimal residual disease by polymerase chain reaction (PCR) or flow cytometry after 2 to 6 weeks of induction is the most precise and useful measure. Augmented therapy has improved outcome for the poor responders to initial treatment. Infants with mixed-lineage leukemia (MLL)–rearranged ALL comprise a very poor-risk group wherein further intensification of chemotherapy causes significant toxicity. Hybrid protocols incorporating drugs effective for acute myeloid leukemia could improve survival, a strategy being tested in international trials. Studies on the biology of MLL-induced leukemogenesis have prompted the development of novel targeted agents, currently under evaluation in clinical trials. Short-term outcomes of patients with Philadelphia chromosome (Ph)–positive ALL have improved significantly by adding tyrosine kinase inhibitors to standard chemotherapy regimens. New agents and methods to overcome resistance are under investigation, and allogeneic stem cell transplantation is recommended for certain subsets of patients, for example those with Ph+ and T-cell ALL with poor early response. Genome-wide interrogation of leukemic cell genetic abnormalities and germline genetic variations promise to identify new molecular targets for therapy.

Construction and Preclinical Evaluation of an Anti-CD19 Chimeric Antigen Receptor

T cells can be engineered to express the genes of chimeric antigen receptors (CARs) that recognize tumor-associated antigens. We constructed and compared 2 CARs that contained a single chain variable region moiety that recognized CD19. One CAR contained the signaling moiety of the 4-1BB molecule and the other did not. We selected the CAR that did not contain the 4-1BB moiety for further preclinical development. We demonstrated that gammaretroviruses encoding this receptor could transduce human T cells. Anti-CD19-CAR-transduced CD8+ and CD4+ T cells produced interferon-gamma and interleukin-2 specifically in response to CD19+ target cells. The transduced T cells specifically killed primary chronic lymphocytic leukemia (CLL) cells. We transduced T cells from CLL patients that had been previously treated with chemotherapy. We induced these T cells to proliferate sufficiently to provide enough cells for clinical adoptive T cell transfer with a protocol consisting of an initial stimulation with an anti-CD3 monoclonal antibody (OKT3) before transduction followed by a second OKT3 stimulation 7 days after transduction. This protocol was successfully adapted for use in CLL patients with high peripheral blood leukemia cell counts by depleting CD19+ cells before the initial OKT3 stimulation. In preparation for a clinical trial that will enroll patients with advanced B cell malignancies, we generated a producer cell clone that produces retroviruses encoding the anti-CD19 CAR, and we produced sufficient retroviral supernatant for the proposed clinical trial under good manufacturing practice conditions.