Peripheral Blood Lymphocyte Count Research Articles

The influence of electrical stimulation on the peripheral immune system under the experimental and clinical conditions

The authors for the first time provide the scientifically-grounded substantiation for the application of electrical stimulation with bipolar pulsed currents for the combined treatment of the patents presenting with acne vulgaris. Experiments on the rabbits have demonstrated the influence of bipolar pulsed currents on the cellular composition of lymph nodes and thereby facilitated the better understanding of certain theoretical aspects of the application of electrical stimulation at large. The clinical study on the application of multi-channel electrical stimulation and microcurrent therapy showed that these physical factors may cause remodeling of immunogenesis in the patents presenting with acne vulgaris manifested as the normalization of the cellular composition of peripheral blood (leukocyte, lymphocyte, T- and B-lymphocyte counts and immunoglobulin levels). These findings confirm the effectiveness of the proposed integrated treatment responsible for the enhancement of the overall resistance of the patents with acne vulgaris. Moreover, this therapeutic modality proved to exert the immunocorrective action and promote the restoration of the adaptive capacity at large. As a result, 80.9% of the patients presenting with acne vulgaris enjoyed prolonged remission of the disease.

Clinical analysis of 71 cases diagnosed first as Pneumocystis carinii pneumonia in AIDS

Objective To discuss the epidemiological and clinical features of patients with AIDS initially diagnosed as Pneumocystis carinii pneumonia(PCP). Methods The retrospective epidemiological survey and clinical study was conducted on the material from 71 patients with AIDS who were initially diagnosed as PCP. Results All cases in this group were admitted to hospital in a critical condition, with fever, dry cough, fatigue, dyspnea and hypoxemia; X- ray and CT showed increased lung markings, thickening, miliary and(or)ground glass- like changes 67 cases(62.0%); most cases had significantly lower CD+4 T lymphocyte and peripheral blood lymphocyte counts, leading to a high death rate. PCP were used sulfamethoxazole therapy,64 cases crued, 7 death. Conclusion PCP in AIDS has clinical and epidemiological characteristics. Paying more attention to epidemiological survey, along with timely confirmation and treatment will be beneficial to improve the prognosis of PCP. Key words: Acquiredimmunodeficiency Syndrome; Pneumonia; Epidemiology

Lymphocyte-to-monocyte ratio predicts survival of patients with hepatocellular carcinoma after curative resection.

To investigate the prognostic value of preoperative lymphocyte-to-monocyte ratio (LMR) in patients with hepatocellular carcinoma (HCC) undergoing curative hepatectomy. Clinicopathological data of 210 hepatitis B virus (HBV)-associated HCC patients who were treated by radical hepatic resection between 2003 and 2010 were retrospectively analyzed. None of the patients received any preoperative anticancer therapy or intraoperative radiofrequency ablation. The diagnosis was confirmed by pathological examination after surgery. Absolute peripheral blood lymphocyte and monocyte counts were derived from serum complete blood cell count before surgery, and LMR was calculated by dividing lymphocyte count by monocyte count. The best cutoff was determined by receiver operating characteristics (ROC) curve analysis. Correlations between LMR levels and clinicopathological features were assessed using the χ(2) test. Survival outcomes were estimated using the Kaplan-Meier method and compared by the log-rank test. Univariate and multivariate analyses were performed to evaluate the prognostic impact of LMR and other clinicopathological factors on overall survival (OS) and recurrence-free survival (RFS), using the Cox proportional hazards model. The optimal cutoff value of LMR for survival analysis was 3.23, which resulted in the most appropriate sensitivity of 55.3% and specificity of 74.7%, with the area under the curve (AUC) of 0.66 (95%CI: 0.593-0.725). All patients were dichotomized into either a low (≤ 3.23) LMR group (n = 66) or a high (> 3.23) LMR group (n = 144). A low preoperative LMR level was significantly correlated with the presence of cirrhosis, elevated levels of total bilirubin and larger tumor size. Patients with a low LMR level had significantly reduced 5-year OS (61.9% vs 83.2%, P < 0.001) and RFS (27.8% vs 47.6%, P = 0.009) compared to those with a high LMR level. Multivariate analyses indicated that a lower LMR level was a significantly independent predictor of inferior OS (P = 0.003) and RFS (P = 0.006). Subgroup analysis indicated that survival outcome was significantly more favorable in cirrhotic patients with LMR > 3.23. However, there were no differences between low and high LMR groups for OS and RFS in non-cirrhotic patients. Preoperative LMR was demonstrated for the first time to serve as an independent prognostic factor in HBV-associated HCC patients after curative resection. Prospective studies with larger cohorts for validation are warranted.

Role of autoantibodies to various Ro60 epitopes in the decrease of lymphocytes seen in systemic lupus erythematosus and primary Sjögren’s syndrome

We investigated the roles of autoantibodies to different Ro60 epitopes in lymphopenia in systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS). We recruited 16 patients with SLE, 14 with pSS, and 10 healthy controls; all were female. Patients had active disease, had not received glucocorticoid or immunosuppressants for at least 3 months, and had positive laboratory tests for autoantibodies against Ro60. Patient peripheral blood lymphocyte (LC) counts were < 1 x 10(9)/L: (0.66 ± 0.12) x 10(9)/L and (0.70 ± 0.16) x 10(9)/L for SLE and pSS groups, respectively (P = 0.511). LCs from each group were cultured in vitro with each of the three immunotoxins (ITs) (AE1-3), which specifically combine with one of the three epitopes (aa482-493, aa310-323, and aa230-241, respectively) on Ro60. The cytotoxicity of each IT to the cultured LCs was measured by the MTT colorimetric method. The relationships between IT cytotoxicity and LC counts were analyzed, and autoantibodies against the three epitopes in patient peripheral blood were detected. All ITs showed cytotoxicity to control LCs; however, AE3 and AE2 showed greater toxicity to LCs from SLE and pSS groups, respectively, and the enhanced cytotoxicity was significantly associated with the respective LC counts (r = 0.653, P = 0.06; r = 0.594, P = 0.025). No difference was found in the prevalence of the autoantibodies between the SLE and pSS groups. These results suggest that autoantibodies to Ro60 might play a pathogenic role in lymphopenia in both SLE and pSS, but the pathogenic mechanisms might differ.

Peripheral Blood Lymphocyte Subsets Counts in Children on Regular Hemodialysis

Background: Hemodialysis (HD) procedure per se as well as disturbances in both innate and adaptive immunity makes HD patients susceptible to infections. Infections are the major cause of morbidity and the second cause of death following cardiovascular events in HD patients. Aim: Was to study lymphocyte subset counts in children with chronic kidney disease on regular hemodialysis in comparison with normal subjects to clarify the abnormalities in cellular immune profile. Patients and methods: The study included 40children with chronic kidney disease on regular hemodialysis and 20 healthy control (HC) children age and sex matched as a control group, they were selected from the pediatric hemodialysis unit and out patients clinic of AL-zahraa hospital ,Al-Azher university during the period from September 2013to June 2014. We examined the number of the peripheral lymphocytes. Also quantification of (T CD3+ & B CD19+) lymphocytes and T cell subsets including T helper CD3+CD4+, T cytotoxic CD3+ CD8+, CD4/CD8 ratio, and NK cells CD3-CD56+ using flow cytometric analysis and correlates their number with clinical and laboratory characteristics. Results: The total peripheral blood lymphocyte count was lower in HD children (2.3 x10³/uL) than HC children (3 x10³/uL), also T lymphocytes CD3+ counts were reduced in HD children (1609.25 ±545.77 / uL) than HC (2114.20± 868.39 /uL), (p=0.008), numbers of CD4+ T cells were not different, but numbers of CD8+ T cells were lower in HD children (600.67±284.92) compared with HC (896.80±573.00) (p < 0.05). Decrease in NK cell counts CD3-CD56+ in HD children (175.35±107.44) in comparison to HC (271.30±169.94). The B lymphocyte count CD19+ was not different in HD children (260±201.6) compared with healthy controls (250.20±122.84). A positive correlation was found between B lymphocytes CD19+ with hemodialysis duration. A negative correlation between NK cell counts and BUN, age and also with patients weight and height were found. Conclusion: The reduced numbers of lymphocyte count, T lymphocytes CD3+counts, CD8+ T cells as well as NK cell in CKD children on regular hemodialysis may favor the frequent occurrence of infections in spit the normal number of the other studied t cell subsets.

Differential effects of ponesimod, a selective S1P1 receptor modulator, on blood-circulating human T cell subpopulations

Ponesimod, a novel selective sphingosine-1-phosphate 1 receptor modulator in the development for the treatment of autoimmune diseases, dose-dependently reduced lymphocyte counts in peripheral blood of healthy subjects. It rapidly and transiently reduced the number of circulating T and B cells, but not natural killer cells. T lymphocyte subsets exhibited differential sensitivities with a maximum decrease from baseline ranging from 67% to 89% following high doses. Naïve T cells were more sensitive than memory T cells. CD4+ T cells were more sensitive than CD8+ T cells or CD4+CD25+ T regulatory cells. The differential effects on specialized T cell subsets may contribute to the immunomodulatory activity of ponesimod. The therapeutic potential of ponesimod has been recently shown in phase II studies of chronic plaque psoriasis and relapsing–remitting multiple sclerosis. Our data suggest that lymphocyte sequestration underlies the therapeutic potential of ponesimod in multiple autoimmune and chronic inflammatory diseases.

The Experience of the International Registry for Chronic Myeloid Leukemia (CML) in Children and Adolescents (I-CML-Ped Study): Pronostic Consideration

▪Aims: An international registry (I-CML-Ped Study)was established to assess epidemiology, management and outcome of CML in the pediatric population.Methods: All national pediatric study groups were invited to include newly diagnosed children and adolescents less than 18 years with CML diagnosed later than January 2000.Results: Since January 2011, 351 patients from 12 countries have been registered. Clinical and biological data at initial diagnosis are available in 278 patients.There was a male preponderance (57%). The median age at diagnosis was 12.4 years (range, 9 months -17.5 years); 6% of the patients were younger than 4 years. At time of diagnosis 92% of the children were in chronic phase, 8% in accelerated phase and 1% in blastic phase according to the European Leukemia Net criteria. The Sokal risk group distribution was: 18% low, 31% intermediate and 51% high risk. The majority of the patients showed a Lansky score of 100 (59%) or 90 (21%). Splenomegaly was present in 76% of patients. The median of the spleen size below the costal margin was 11 cm (range: 1 to 25 cm). The median of the leukocyte count was 235x109/L (range: 5 to 1038). Additional chromosomal abnormalities in Ph-positive cells were observed in 6 % of the patients. The BCR-ABL transcript type was available in 227 patients: b3a2 54%, b2a2 38%, b3a2-b2a2 6% and b2a3 2%. The median follow-up time is 39 months (range, 0.5-161). Eight deaths were recorded. The estimated overall survival rate at 60 months is 95% (95%CI 89-97). Irrespective of treatment and follow-up, 124 (73%) of 169 assessable patients for cytogenetic response achieved complete cytogenetic response (CCyR). Exploratory analyses were performed in newly diagnosed patients regarding clinical and biological factors influencing the achievement of CCyR 12 months after starting imatinib. In univariate analyses, the Eutos score, the spleen size, the hematocrit level, the lymphocyte count and immature cells in peripheral blood, the percentage of granulocytes and monocytes in the marrow were identified as potential prognostic factors. However, only the percentage of the granulocytes in the marrow was identified as independent factor of achievement of CCyR at 12 months in multivariate analysis. Data collection and quality control regarding molecular assessment are ongoing.Conclusion: The data indicates that children and adolescents with CML presented with clinical and biological differences compared to adult patients with CML. Identification of prognostic factors is needed to optimize the strategy in the pediatric population.Acknowledgment: The I-CML Ped study is supported by an unrestricted grant from Novartis Pharmaceutical Company DisclosuresNo relevant conflicts of interest to declare.

Absolute Lymphocyte Count As an Independent Prognostic Factor of IPI and NCCN-IPI in DLBCL Patients

Background: Previous studies have shown that the absolute lymphocyte count (ALC) in peripheral blood at diagnosis may be an independent prognostic factor of IPI for patients with diffuse large B-cell lymphoma (DLBCL). In the rituximab era, the U.S. National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) was developed to improve the risk stratification of DLBCL in comparison to the existing IPI. Therefore, the aim of this study was to clarify the impact of ALC at diagnosis on event free survival (EFS) and overall survival (OS) on analysis performed with factors included in NCCN-IPI.Patients and methods: We retrospectively reviewed the ALC of 413 patients with newly diagnosed DLBCL treated with R-CHOP at our hospital between January 2005 and March 2013. Primary central nervous system lymphoma patients were excluded from this study. ALC was determined in all patients from complete blood count with differential white blood count at the time of diagnosis, and prior to therapy administration. EFS and OS were estimated according to the Kaplan-Meier method. Multivariate analysis was performed with the proportional hazard Cox model.Results: The median ALC was 1.2x10E9/L (range, 0.06-9.0). We set an ALC cut-point at 1.0x10E9/L based on previous studies. The median follow-up duration was 40 months. Baseline characteristics according to ALC (<1.0x10E9/L[n=145] and >1.0x10E9/L[n=268]) are summarized in Table1. Patients with ALC<1.0x10E9/L had a significantly poorer EFS and OS than patients with ALC>1.0x10E9/L (5-year EFS, 37.0% versus 68.9%, p<0.001; 5-year OS, 46.3% versus 80.0%, p<0.001). On multivariate analysis performed with factors included in IPI and NCCN-IPI, ALC remained an independent predictor of EFS (IPI: hazard ratio [HR] 1.95; 95% confidence interval [CI] 1.43-2.68; p<0.001, NCCN-IPI: HR 1.94; 95%CI 1.42-2.65; p<0.001) and OS (IPI: HR 2.35; 95%CI 1.61-3.42; p<0.001, NCCN-IPI: HR 2.29; 95%CI 1.57-3.33; p<0.001) (Table2). Importantly, within the poor R-IPI group, ALC distinguished patients with different 5-year EFS (24.4% versus 50.4%, p<0.001) and OS (35.7% versus 65.7%, p<0.001). For the high NCCN-IPI group also, ALC distinguished patients with different 5-year EFS (14.8% versus 39.8%, p<0.01) and OS (17.5% versus 54.5%, p<0.001) (Figure1).Conclusions: According to our results, ALC<1.0x10E9/L is an adverse prognostic factor and independent of IPI and NCCN-IPI. ALC might be more successful in identifying high-risk patients in which IPI and NCCN-IPI analysis was unrevealing. Our results suggest that other therapeutic strategies may be more effective in high-risk patients with DLBCL. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Peripheral Absolute Neutrophil, Mmonocyte and Lymphocyte Counts and Clinical Ooutcome in Diffuse Large B-Cell Lymphoma

BackgroundBaseline levels of peripheral absolute neutrophil (ANC), lymphocyte (ALC) and monocyte (AMC) counts have prognostic value in solid tumors. There are limited data on their impact on outcomes in patients with diffuse large B-cell lymphoma (DLBCL). The present study is to confirm whether the baseline levels of peripheral ANC, ALC and AMC can predict outcomes in patients with newly diagnosed DLBCL.Patients and MethodsData from lymphoma database at the University of Texas MD Anderson Cancer Center (UTMDACC) were used. The correlation of baseline ANC, AMC and ALC with progression-free survival (PFS) and overall survival (OS) were analyzed in 817 newly diagnosed previously untreated patients with DLBCL who received frontline treatments at UTMDACC between 2000-2011. Kaplan-Meier method was used for time-to event analysis including PFS and OS. Multi Cox proportional hazards model were fitted to evaluate important patient characteristics on the time-to event endpoints.ResultsMedian ages at diagnoses were 58 years (range, 18 to 91 years). Seventy-nine percent of patients received conventional immunochemotherapy(CHOP-R) while 21 % of patients had intensive immunochemotherapy (HyperCVAD-R or R-EPOCH) or intensive chemotherapy(n=5; HyperCVAD or R-EPOCH) due to CD 20 negative; two patients received radiation therapy only. IPI low, intermediate and high were 38%, 46% and 16%, respectively. Overall response rate was 98% with a complete remission rate of 73%. Patients with abnormal ANC (> 7.3 X109/L) had an inferior PFS (P<0.001) and OS (P<0.001) compared to patients with ANC ≤ 7.3 X109/L; patients with abnormal ALC (< 1.0 X109/L) had significant short PFS (P<0.001) and OS (P<0.001) compared to patients with ALC ≥ 1.0 X109/L; similarly, patients with abnormal AMC (> 0.7X109/L) had inferior PFS (P< 0.001) and OS (P< 0.001) compared to patients with AMC≤ 0.7 X109/L. Multivariate analysis identified abnormal ALC and AMC as an independent adverse prognostic factors for PFS (hazard ratio [HR] 1.39 and 1.49, respectively; P<0.016 and P=0.004, respectively) and abnormal ANC and AMC as an independent adverse prognostic factors for OS (P=0.017 and P=0.024, respectively; HR 1.56 and 1.42, respectively).ConclusionsAn abnormal Neutrophil, lymphocyte and monocyte counts in peripheral blood are independent, poor prognostic factor for PFS and OS in patients with DLBCL. Incorporation of these immunological markers into IPI prognostic model may improve outcome prediction. DisclosuresNo relevant conflicts of interest to declare.

P050 - Efficacy of lower fingolimod doses in the treatment of MS

Background Oral fingolimod is a sphingosine-1-phosphate-receptor modulator that prevents the egress of lymphocytes from lymph nodes. Fingolimod reduces relapses and delays disability progression in patients with relapsing forms of multiple sclerosis (MS). Elevation of liver function tests (LFTs) and reduction in peripheral-blood lymphocyte counts were among the most common serious adverse events reported in Phase II, Phase III, and extension studies. Objective To describe seven patients in whom fingolimod dose was reduced to every other day (n=5) or every third day (n=2) due to increase LFTs>3× ULN (n=2) or decrease lymphocyte count? 0.2×109/L (n=5). Results None of the 7 patients developed clinical relapses after decreasing fingolimod dosage, but one patient had new lesions on brain and spine MRIs. In all patients dose reduction resulted in improvement of the laboratory abnormalities. Conclusion This is the first report of reduced fingolimod dosing in the treatment of MS. Our results suggest that every other or every third day dosing can control disease activity satisfactorily in patients with multiple sclerosis and lymphopenia or elevated LFTs.

T-cell prolymphocytic leukemia frequently shows cutaneous involvement and is associated with gains of MYC, loss of ATM, and TCL1A rearrangement.

T-cell prolymphocytic leukemia (T-PLL) is a rare aggressive mature T-cell leukemia with frequent cutaneous presentation, which has not been well characterized. Among the 25 T-PLLs diagnosed between 1990 and 2013 at our institution, 32% (8/25) showed cutaneous manifestations, presenting as rash, purpura, papules, and ulcers. The skin biopsies showed leukemia cutis with perivascular and periadnexal irregular, small to medium-sized lymphoid infiltrates without epidermotropism. The lymphoid infiltrates were composed of mature CD4+ T cells expressing other T-cell antigens, and a subset (48%) showed dual CD4+/CD8+ coexpression. Higher median absolute peripheral blood lymphocyte count (43.0 vs. 13.0 k/mm; P=0.031) and elevated lactate dehydrogenase levels (P=0.00018) at the time of diagnosis were significantly associated with T-PLLs with skin involvement compared with those without. The extent of bone marrow involvement (P=0.849) and overall survival (P=0.144) was similar in the 2 groups. Fluorescence in situ hybridization or karyotype revealed frequent gains of MYC (67%; n=9), loss of ATM (64%; n=11), and TCL1A rearrangement or inversion 14q (75%; n=12). Gains of TCL1A was also seen (78%; n=9), including in some cases that had concurrent TCL1A rearrangement, whereas TP53 loss was less common (30%; n=10). No correlation was seen between the immunophenotype and morphology versus the presence or absence of skin involvement. These data suggest that cutaneous involvement by T-PLL is relatively common and often associated with significant peripheral blood involvement. The frequent MYC, ATM, and TCL1A alterations identified support that these genes are integral to the pathogenesis of T-PLL.

Serum apelin-12 in bronchial asthma

Aim. To evaluate the serum level of apelin-12 in patients with bronchial asthma. Methods. 11 healthy controls and 29 patients with bronchial asthma were examined (among them 19 with atopic asthma and 10 with non-atopic asthma). Serum apelin-12 levels were measured by enzyme immunoassay (ELISA, MyBioSource, USA). SPSS (Statistical Package for the Social Science, V. 13) for Windows was used for statistical analysis. Results. The phenomenon of low apelin-12 serum levels was revealed in patients with bronchial asthma. The most significant decrease of apelin-12 levels was noticed in atopic bronchial asthma. The apelin-12 levels were significantly correlated with a number of indices of bronchial obstruction. Apelin-12 levels correlated with a number of indicators characterizing the grade of bronchial obstruction. Moreover, low apelin-12 levels were suggesting bronchial obstruction. Besides, significant reverse correlation of apelin-12 levels and allergic sensitization was revealed. In addition, statistically significant relation between apelin-12 serum level and peripheral blood eosinophil and lymphocyte counts was revealed. There was an inverse association between apelin-12 serum level and peripheral blood neutrophil count. Conclusion. Literature analysis and our own results allow us to suppose the possible protective role of apelin-12 in bronchial asthma flare. Further studies of apelin-12 and of other isoforms of adipokines are needed for better understanding of the disease.

Identification of splenic marginal zone lymphoma from B lymphoproliferative disorders by flow cytometry

The splenic marginal zone lymphoma (SMZL) is a relatively rare chronic B lymphoproliferative disease, which primarily manifest increase of peripheral blood lymphocyte count and/or scale, and splenomegaly, while the peripheral superficial lymph nodes are often not swollen. Therefore, the splenectomy are usually needed to confirm the diagnosis, but the majority of patients could not accept such management, resulting in early difficult diagnosis. This study was purposed to explore the more prior way for diagnosis based flow cytometry (FCM). Six patients with suspected diagnosis of SMZL were used as research objects, 10 healthy bone marrow donors and 10 cases of chronic lymphocytic leukemia (CLL), 3 cases of hairy cell leukemia (HCL), 3 cases of lymphatic plasma cell lymphoma/Waldenströ's macroglobulinemia (LPL/WM) were selected as control. The immunophenotype of bone marrow cells were analyzed and compared by FCM using a panel of antibodies including CD45, CD5, CD10, CD19, CD20, CD22, CD23, CD25, CD103, CD11c, CD123, κ,λ, Cyclin D1, and combined with bone marrow cell morphology. The results indicated that 6 cases of suspected SMZL showed a large increase of lymphocytes and splenomegaly. Because absence of peripheral lymphadenopathy, 6 patients did not suffer from lymph node biopsy, only 1 patient underwent diagnostic splenectomy. The immunophenotypes of bone marrow in patients and controls were analyzed by FCM, as a result, except for the healthy donors, varying degrees of abnormal mature B cell clones were found in bone marrow of all patients, and the further differentiation from other B-cell tumors was performed through CD5, CD10 expression and combination with other B-cell phenotype. All 6 cases of SMZL patients expressed CD19(+) and CD20(+), but CD10 expression was negative, 4 patients expressed CD5(-), 2 patients expressed CD5(+). The expressions of CD23, CD38, ZAP-70, CD11c, CD103, CD123, Cyclin D1 were negative. The morphological examination of bone marrow cells showed velutinous abnormal lymphocytes. Combined with clinical characteristics, 6 patients were diagnosed as SMZL, 1 patient suffered from splenectomy because of concurrent hypersplenism, and this postoperative pathologic examination confirmed the patient with SMZL. Ten cases of CLL mainly expressed CD5, CD23; 3 cases of HCL had more typical morphology of "hair like" in addition to CD11c, CD103 and CD123 positive; 3 cases of LPL/WM had significantly increased light chain restriction expression, IgM, plasmacytoid lymphocytes. It is concluded that the FCM immunophenotype analysis can be used as a powerful tools for clinical diagnosis of SMZL.

Correlation of peripheral blood lymphocyte counts with outcome and histopathologic features of lymphocytic inflammation in patients with stage II colorectal carcinoma.

475 Background: The prognostic importance of the local antitumor immune response in colorectal carcinoma (CRC) is well established. Recent studies suggest that systemic antitumor immunity may also impact on survival. This study aimed to determine the relationship between peripheral blood lymphocyte counts (PBLCs), reactive features in regional lymph nodes, local tumoral/peri-tumoral lymphocytic inflammation and survival in stage II CRC. Methods: Resection specimens from 185 patients with stage II CRC were assessed for peri-tumoral lymphocytic inflammation, tumor-infiltrating lymphocytes, lymph node yield, lymph node size, and reactive changes in individual lymph nodes. Reactive changes were scored semi-quantitatively and given a reactivity [R] score of 1-4 based on the presence and size of germinal centers. Pre-operative PBLCs and neutrophil counts were recorded. Results: Lower peripheral lymphocyte counts were associated with advanced age (p = 0.016), T4 disease (p = 0.006), venous invasion (p = 0.006), lymph node yields &lt;12 (p = 0.024), smaller mean nodal diameters (p = 0.021), absence of tumor-infiltrating lymphocytes (p = 0.002) and fewer highly-reactive lymph nodes (R score 4) per case (p = 0.032). Cox regression analysis revealed that advanced age (Hazard Ratio, 1.03 per year [95% confidence interval, 1.01-1.05]; p = 0.015) and higher peripheral lymphocyte counts (HR, 0.52 per 1x109cells/L [95% CI, 0.31-0.86]; p = 0.011) were independently predictive of disease-free survival. Neutrophil counts were not associated with outcome. A higher neutrophil to lymphocyte ratio was associated with worse survival on univariate, but not multivariable analysis. Conclusions: The PBLC is an independent predictor of survival in stage II CRC, and is significantly associated with the immune status of regional lymph nodes and the local lymphocytic immune response.

Pre-treatment Haemoglobin and Peripheral Blood Lymphocyte Count as Independent Predictors of Outcome in Carcinoma of Cervix

AimsTo evaluate pre-treatment haemoglobin and peripheral blood lymphocyte (PBL) counts as predictors of treatment outcome in cervix carcinoma treated with radical chemoradiation. Materials and methodsPre-treatment PBL counts and haemoglobin concentrations were retrieved from full blood count examinations from 111 patients who received concurrent chemoradiotherapy. Overall survival and relapse-free survival were obtained using the Kaplan–Meier method by ranking the data by median haemoglobin and PBL, singly and then in association. Their independence and significance as predictors of outcome were analysed using the Cox proportional hazard model. ResultsSurvival rates were significantly higher in patients whose haemoglobin level or PBL counts were at or above the corresponding median value. At 5 years, rates of overall survival were 77% versus 41% (P = 0.0003) and 75% versus 42% (P = 0.002), when dichotomised around median haemoglobin and PBL, respectively. In multivariate and univariate analyses, both PBL and haemoglobin were independent and significant predictors for risk of death and relapse. Their predictive power was dramatically enhanced when the data were stratified into four groups by associating patients with haemoglobin ≥ median or < median with those whose PBL was ≥ or < median. ConclusionBaseline PBL and haemoglobin seem to be strong, independent predictors of treatment outcome in carcinoma of the cervix, particularly if patient response is ranked using the predictors simultaneously. The hypothesis needs to be tested and, if confirmed, the markers should be used in combination to identify those at greater risk of failure who may benefit from additional therapy, with further validation in prospective trials offering treatment modification.

Prediction of and prophylaxis against Pneumocystis pneumonia in patients with connective tissue diseases undergoing medium- or high-dose corticosteroid therapy.

We performed a retrospective analysis to establish a statistical model for the prediction of Pneumocystis pneumonia (PCP) in patients with connective tissue diseases (CTD) undergoing medium- or high-dose corticosteroid therapy, to identify independent risk factors for PCP and to evaluate the efficacy of the prophylactic use of trimethoprim-sulfamethoxazole (TMP/SMX) against PCP. One hundred and twenty-four patients who were receiving the equivalent of or more than 30 mg/day of prednisol-one (PSL) were classified into two groups according to the presence (prophylaxis group, n = 46) or absence (nonprophylaxis group, n = 78) of prophylactic TMP/SMX. We developed a statistical model that was suitable for predicting the development of PCP using a logistic regression analysis. The initial steroid dosage, decreased peripheral blood lymphocyte counts at 2 weeks (<500/microl), and usage of immunosuppressant during 2 weeks after the institution of PSL (>or=30 mg/day) were found to independently contribute to the development of PCP. Finally, in the patient group with a defined risk for PCP, a significant prophylactic effect of TMP/SMX was demonstrated. We recommend the prophylactic use of TMP/SMX for patients with CTD undergoing medium- or high-dose corticosteroid therapy who are determined to have a high risk of developing PCP.

Serum adenosine deaminase activity in cutaneous anthrax.

BackgroundAdenosine deaminase (ADA) activity has been discovered in several inflammatory conditions; however, there are no data associated with cutaneous anthrax. The aim of this study was to investigate serum ADA activity in patients with cutaneous anthrax.Material/MethodsSixteen patients with cutaneous anthrax and 17 healthy controls were enrolled. We measured ADA activity; peripheral blood leukocyte, lymphocyte, neutrophil, and monocyte counts; erythrocyte sedimentation rate; and C reactive protein levels.ResultsSerum ADA activity was significantly higher in patients with cutaneous anthrax than in the controls (p<0.001). A positive correlation was observed between ADA activity and lymphocyte counts (r=0.589, p=0.021) in the patient group.ConclusionsThis study suggests that serum ADA could be used as a biochemical marker in cutaneous anthrax.

Oncogenic Insult In Bone Marrow Endothelial Cells Causes Hematopoietic Abnormalities

Bone marrow endothelial cells (BMECs), the cells forming bone marrow vasculature, have been implicated as a critical constituent of bone marrow microenvironment essential for the maintenance of normal and malignant hematopoietic stem cells. They are a part of BM niche regulating the self-renewal and differentiation of hematopoietic stem cells (HSCs) and the homing and progression of pre-leukemia and leukemia cells. It has been realized that pathological angiogenesis such as increased bone marrow microvessel density is associated with a spectrum of human diseases including MDS, MPD and leukemia. Recent discovery of oncogenic mutations of BCR-ABL and JAK2V617F in BMECs and spleen ECs primes the hypothesis that pathological angiogenesis from oncogenic insults may promote or facilitate abnormal hematopoiesis leading to the development and maintenance of blood malignancies. Clinical studies have linked germline mutations of Ras signaling components (e.g. NF1, SHP2, CBL and K-RAS) with JMML, a subgroup of MPD/MDS that is difficult to cure and for which bone marrow transplantation often results in relapses and graft failure. Studies in animal models have shown that activation of oncogenic K-Ras in hematopoietic stem cells could partially recapitulate the symptoms of these MPD/MDS patients, suggesting a HSC intrinsic role of oncogenic transformation. It remains to be seen if deregulated K-Ras signaling in the BM microenvironment, BMECs in particular, may also contribute to the hematologic abnormalities and disease progression, and whether pathologicial BMECs provide an addition therapeutic opportunity to limit transplant-associated relapse. To this end, we recently established a new mouse model that allows endothelial lineage-specific, inducible genetic manipulation of BMECs in adult animals in vivo and a cell culture system to select, expand and functional assay freshly isolated murine BMEC activities in vitro. A strain of Tie2-CreER transgenic mice crossed with loxP-stop-loxP fluorescence reporters (EGFP or Td-tomato) is sensitive to Tamoxifen induction in specifically turning on Cre activity in endothelial cells in 4-8 week old animals with over 80% efficiency in BMECs. Our culture conditions select and expand mouse primary BMECs in a large scale without immortalization or genetic manipulation, and the resulting BMECs retain endothelial lineage markers and are fully functional in 2-demensional and 3-demensional capillary formation assays as well as Matrigel implant plug assay. Using this inducible model, we generated Tie2CreER/LSL-K-Ras G12D mice, to specifically turn on K-Ras G12D in endothelial cells to test if an oncogenic K-Ras insult in EC compartment can affect hematopoiesis. Three consecutive Tamoxifen injections to the mice readily activated K-Ras G12D expression in the BMECs, but not in the blood lineages. The mice bearing endothelial K-Ras G12D died 4-6 months after Tamoxifen induction. An analysis of the K-Ras G12D mice revealed that the oncogenic insult caused a significant change in BM vascular structure and abnormal hematopoiesis. K-Ras G12D mutation in the ECs led to pathological angiogenesis in the bone marrow as manifested in the K-Ras G12D mice an increased colony-forming unit EPC activity, drastically increased vessel permeability, and increased BMEC proliferation. The pathological angiogenesis in the bone marrow was associated with abnormal blood phenotypes in K-Ras G12D mice, showing a significant increase of WBC and lymphocyte counts in peripheral blood, splenomegaly, severe anemia, and a significant increase of CFU-C activity in the blood and spleen. Ongoing transplant experiments will address whether the oncogenic K-Ras expression in endothelial cells is sufficient to fully promote blood cell transformation. In parallel, the oncogenic K-Ras expressing BMECs in primary culture are being examined to determine whether abnormal Ras signaling including RAF-MEK and/or PI3K-AKT pathways are involved in mediating BMEC proliferation and angiogenesis. Our study suggests a novel concept that pathologic bone marrow microenvironment, BMECs in particular, can play an important role in blood cell transformation, and implies that rational targeting of oncogenic pathways in the BMECs may provide an additional therapeutic avenue for blood malignancies, besides direct suppression of abnormal hematopoietic cells, to restore normal hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Poly [ADP-Ribose] Polymerase 1 (PARP-1) Expression Before and After Immunochemotherapy In Patients With B Chronic Lymphocytic Leukemia

IntroductionPoly [ADP-ribose] polymerase 1 (PARP-1) has a central role in the repair of single-stranded DNA (ssDNA) breaks, thus protecting the cell from genomic instability. This enzyme is a promising target in anticancer treatment. The aim of the present study is to investigate the role of PARP1 in B chronic lymphocytic leukemia (B-CLL) patients pre- and post- treatment. Patients and MethodsBlood samples were collected from B-CLL patients before treatment and following 3 cycles of immunochemotherapy (chosen by the treating physician according to common clinical practice). Quantification of PARP1-mRNA levels was performed by a SYBR-green based PCR performed on BIORADCFX96 (BIORAD). The results were expressed in correlation to the mRNA levels of the endogenous housekeeping gene of beta actin (ACTB). The detection of the gene product was performed by Western Blot using anti-PARP1 antibody (clone 47-258; Calbiochem). We detected the 70 KD protein segment that results from the in vivo fractionation of PARP1 by caspase-3. The protein bands were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences) and quantitated by densitometry using the gel analysis software ImageJ. We correlated the PARP1 protein levels to those of a group of healthy volunteers. We also studied the differences between PARP1-mRNA and protein levels before and after treatment and correlated them to clinical response. The related samples Wilcoxon Signed Rank test (IBM SPSS statistics, version 19.0) was used for the statistical analysis of the results. ResultsSamples were obtained from 24 B-CLL patients before treatment and from 15/24 following 3 cycles of immunochemotherapy. The patients' characteristics are shown in Table 1. Pre-treatment PARP1 protein levels were similar to those of 15 healthy volunteers (p=0.426). No statistically significant difference was noted between the PARP1-mRNA and protein levels following treatment (p=0.507 and 0.650 respectively) (Table 1). Multivariate analysis did not reveal statistically significant differences in the mRNA and protein levels in correlation to the stage of disease, the peripheral blood lymphocyte count, the LDH levels, the response to treatment and the overall prognosis of the patients. ConclusionsThe expression of PARP1 in patients with B-CLL seems to be comparable to that of healthy persons. We could not distinguish patients displaying reduced levels of PARP1, as previously reported by others. Moreover, PARP1 expression is not affected by immunochemotherapy, irrespective of stage and response to treatment. Thus, this molecule seems not to participate in the mechanisms involved in DNA repair and apoptosis that are impaired in B-CLL. The study is still ongoing with the addition of more subjects. Disclosures:No relevant conflicts of interest to declare.

The Presence Of Normal B and T Lymphocytes In Bone Marrow At Diagnosis Predicts Overall and Event Free Survival In Childhood B- Lineage Acute Lymphoblastic Leukaemia

IntroductionThe absolute lymphocyte count (ALC) in peripheral blood has been shown to be a prognostic indicator in both adult and paediatric leukaemia. (Hudson et al 2004, Rabin et al 2012)The ALC is far easier and cheaper to measure than minimal residual disease in both developed and developing countries [1]. It is not known which lymphocytes in peripheral blood are important in predicting outcome or the mechanism for this. AimsWe aimed to investigate the presence and subtype of normal lymphocytes present in the bone marrow at diagnosis in patients with B lineage acute lymphoblastic leukaemia and correlate with clinical outcome. We collected retrospective patient data from 101 patients at Alder Hey Children's hospital diagnosed with B lineage ALL between 2002 and 2008. We aimed to identify normal non-tumour B and T lymphocytes present at diagnosis in bone marrow and correlate their presence with event free(EFS) and overall survival(OS). MethodsPatient data was collected from 101 patients' records (including clinical prognostic information cytogenetics and MRD status). All patients had at least 5 years follow up. Diagnostic flow cytometry list mode data files were analysed using WinMDI 2.9 software to determine the tumour populations, and proportions of non-tumour lymphocytes using CD20 and CD7 monoclonal antibodies, which were included in the original diagnostic panel. Proportions of these normal cell populations were correlated with patient outcome. One patient was excluded from the normal B cell analysis due to a high CD20 tumour expression. ResultsThe analysis of CD7 lymphocytes is shown in figure 1. [Display omitted] We analysed CD7+ cells as a % of the non-leukaemia cells. Using a threshold of 29.1% those with high CD7 cell numbers OS was 96.8% below the threshold OS was 85.3% (p=0.0017). Event free survival was 93.6%v 82.3% in those with high v low numbers (p=0.004) see figure 1.The mean proportion of CD20+ cells was 3.86% (SD +/- 7.66)% in the alive patients (n=94) and 1.21 (SD +/-0.75)% in deceased patients (n=6). Using a threshold value of 2.4% the overall survival in those with high CD20 cell numbers was 100.00% v 89.65% in those with low numbers, (p-= 0.03).Similarly a strong trend was observed with CD20 numbers and event-free survival in patients above or below 2.7%, EFS was 97.1% and 87.9% respectively p= 0.129. Conclusion/DiscussionWith modern therapy fortunately most children survive their leukaemia. However there is a group of patients that remain not possible to cure with current chemotherapy approaches.We present that the presence of normal CD7+ and CD20+ lymphocytes in the bone marrow at diagnosis are strongly associated with overall and event free survival. The mechanism of this effect is not known. We postulate that within these cell populations, that we have identified, is a specific immune response to the individuals own leukaemia. If this is the case these normal lymphocytes could be harvested and used as a potential individualised therapy. Disclosures:No relevant conflicts of interest to declare.