β2M Levels Research Articles

Expression stability of putative reference genes in equine endometrial, testicular, and conceptus tissues

BackgroundQuantitative RT-PCR data are commonly normalized using a reference gene. A reference gene is a transcript which expression does not differ in the tissue of interest independent of the experimental condition. The objective of this study was to evaluate the stability of mRNA expression levels of putative reference genes in three different types of equine tissue, endometrial, testicular, and conceptus tissue.FindingsThe expression stability of four (uterine tissue) and six (testicular and conceptus tissue) was assessed using descriptive data analysis and the software programs Normfinder and geNorm. In uterine samples, 18S showed the largest degree of variation in expression while GAPDH, B2M, and ACTB were stably expressed. B2M and GAPDH were identified as the most stably expressed genes in testicular samples, while 18S showed some extent of regulation between samples. Conceptus tissue overall was characterized by very low variability of the transcripts analyzed with GAPDH, YWHZ, and 18S being the most stably expressed genes.ConclusionsIn equine endometrium, GAPDH, B2M, and ACTB transcript levels are equally stable, while 18S is less stably expressed. In testes and associated structures, B2M and GAPDH are the transcripts showing the least amount of variation, while in conceptus tissue GAPDH, YWHZ, and 18S were identified as the most suitable reference genes. Overall, transcripts analyzed in conceptus tissue were characterized by less variation than transcripts analyzed in uterine and testicular tissue.

BAFF AND TACI mRNA expression are increased in very early rheumatoid arthritis patients

BackgroundB cells play several important roles in rheumatoid arthritis (RA). Previous studies by our group have demonstrated that very early rheumatoid arthritis (VERA) patients have disturbances in peripheral blood memory...

β2-microglobulin, a novel biomarker of peripheral arterial disease, independently predicts aortic stiffness in these patients

Arterial stiffness is a prominent feature of vascular ageing and strongly predicts cardiovascular and total mortality. The β2-microglobulin, (β2M) a newly identified biomarker of peripheral arterial disease (PAD), is related to renal insufficiency, inflammatory and neoplastic diseases, but may also play a role in vascular dysfunction. However, the relationship between arterial stiffness and β2M has not been previously studied in patients with atherosclerosis. In the present study we examined a possible association between β2M and arterial stiffness in patients with PAD and in healthy subjects. Plasma β2M levels and parameters of arterial stiffness such as aortic pulse wave velocity (aPWV) and augmentation index (AIx) were measured in 66 patients with PAD and in 66 apparently healthy subjects. Plasma levels of β2M, aPWV and AIx were significantly increased in patients with PAD compared with controls (1858.1 ± 472.8 vs 1554.5 ± 277.9 μg/L, p < 0.001; 9.9 ± 2.2 m/s vs 7.6 ± 1.6 m/s, p < 0.001; 28 ± 8 vs 14 ± 11%, p < 0.001; respectively). There existed significant correlation between aPWV and β2M for the patient group (R = 0.47; p < 0.001), but not for the controls (R = 0.14; p = 0.26). In multivariate analysis, β2M remained independently associated with aPWV, fetuin-A, age and glomerular filtration rate in patients (R2 = 0.5, p < 0.001). We found no relationship between β2M and AIx in either group. We demonstrated that among patients with PAD elevated plasma β2M levels were associated with higher aortic stiffness irrespective of cardiovascular disease risk factors. These data suggest that β2M may influence the pathogenesis of aortic stiffness in atherosclerosis.

Beta-2 microglobulin as an immunological marker to assess the progression of human immunodeficiency virus infected patients on highly active antiretroviral therapy

BackgroundAcquired immune deficiency syndrome (AIDS) defines the end stage of Human immunodeficiency viral (HIV) infection before the introduction of highly active antiretroviral therapy (HAART). This study was carried out to assess the serum β-2 microglobulin (B2M) as a marker for progression of HIV infected patients undergoing HAART. MethodsBlood samples were collected from 50 subjects of HIV infected patients residing at semi urban area undergoing treatment at Chellam Hospital, Salem, India. Twenty five age-matched healthy subjects were taken as control group. Serum B2M level was measured by using enzyme immunoassay, absolute CD4 and CD8 counts were carried out by single platform (SP) technology using a flow cytometer. Viral RNA was measured by real time PCR. The serum LDH level and total WBC count were also measured. ResultsOur result showed a statistically high significant increase in B2M in HIV patients on HAART non complaint group whereas absolute CD4, CD8 count, CD4/CD8 ratio and WBC count were decreased significantly when compared to control and HAART complaint group. Statistically a significant negative correlation was observed between B2M and absolute CD4, CD8 count, CD4/CD8 ratio and WBC count. B2M showed a significantly positive correlation with viral RNA and LDH values. ConclusionsThe increase of B2M and reduced absolute CD4, CD8 count, CD4/CD8 ratio and WBC count in HIV patients on HAART non complaint group may have a contributory role in the immune progression of HIV with interruption of HAART. B2M plays an important role in the diagnosis of HIV and might indicate HIV progression.

The Impact of Advanced Age According to IPSSWM Cut-Off on the Outcome of Symptomatic and Asymptomatic Waldenström's Macroglobulinemia at Diagnosis

Advanced age is one of the variables more frequently considered to be associated with an adverse prognosis in Waldenström's macroglobulinemia (WM). In a series of 238 symptomatic and asymptomatic WM patients, we retrospectively identified an age cut-off distinguishing two groups of patients with different outcome in terms of overall survival (OS), disease-specific survival (DSS) and treatment-free survival (TFS). Although for the OS the best cut-off was identified at 65 years with shorter OS for elderly patients, no difference was detected in terms of DSS between the two groups. Furthermore, patients over 65 years showed a longer TFS compared with patients under 65 years. Clinical and laboratory disease characteristics did not significantly differ between the two groups of patients except for β2M level. Therefore, the poorer survival of patients over 65 years at diagnosis should probably be attributed to the higher number of no disease-related deaths and is independent from WM.

Experience with rituximab immunotherapy as an early intervention in patients with Rai stage 0 to II chronic lymphocytic leukemia

Management of asymptomatic early stage chronic lymphocytic leukemia (CLL) centered on expectant surveillance for active disease warranting chemotherapy. In CLL, elevated serum β-2 microglobulin (β2M) levels were associated with more rapid disease progression and shorter survival (OS). An early intervention trial was designed to assess response, time-to-progression (TTP), and OS after immunotherapy with standard-dose rituximab (375 mg/m² intravenously weekly for 8 consecutive weeks) in 34 asymptomatic untreated early stage CLL with β2M level ≥ 2 mg/dL. Long-term follow-up and results are reported. The overall response rate in 34 patients was 82% (9% complete [CR]), median TTP in the 28 responders was 23 months, the median time to subsequent treatment was 43 months, and the 8-year OS rate was 74% (median follow-up, 102 months). Early treatment with rituximab was well tolerated and safe. Further studies are needed to determine if this intervention can decrease CLL-related morbidity and mortality.

Urinary β-2-microglobulin as an alternative marker for fetal inflammatory response and development of bronchopulmonary dysplasia in premature infants

To evaluate the clinical utility of urinary β-2-microglobulin (B2M) at birth, an alternative to proinflammatory cytokines, as an indicative marker of fetal inflammatory response and subsequent higher risk of bronchopulmonary dysplasia (BPD) in premature infants. The relationship between urinary B2M at birth and the occurrence of BPD was examined in 96 premature infants with a description of perinatal backgrounds. Constructing a receiver-operating characteristic curve to determine the cutoff value of urinary B2M at birth for the development of BPD, a multivariate logistic regression analysis was performed to evaluate whether elevated urinary B2M at birth can be used as a predictor of BPD. BPD was diagnosed in 34% (33/96) of the infants. Neonates with BPD had a significantly higher occurrence rate of chorioamnionitis and greater levels of median urinary B2M at birth than did those without BPD. The selected cutoff value of urinary B2M at birth correlated with the development of BPD, even after adjusting for gestational age and other confounding factors. Elevated urinary B2M levels at birth can be used as an alternative marker of fetal inflammatory response and subsequent higher risk of BPD in premature infants.

Phase III Intergroup Study of Lenalidomide Versus Placebo Maintenance Therapy Following Single Autologous Hematopoietic Stem Cell Transplantation (AHSCT) for Multiple Myeloma: CALGB 100104

Phase III Intergroup Study of Lenalidomide Versus Placebo Maintenance Therapy Following Single Autologous Hematopoietic Stem Cell Transplantation (AHSCT) for Multiple Myeloma: CALGB 100104

Flow Cytometric Phenotyping and Analysis of CD8 Regulatory and Suppressor Cells In Multiple Myeloma

AbstractAbstract 2947In solid tumors and hematological malignancies, elevated proportions of CD4 regulatory (CD4 Tregs) cells were reported and the function was also documented well. In the context of CD8 regulatory cells (CD8 Tregs) existence has been revealed in various solid tumors including colorectal, prostate and ovarian cancers. In hematological malignancies or multiple myeloma (MM) the presence of CD8 Tregs are yet to be revealed. To unveil and understand the existence of CD8 Tregs, we characterized and quantified CD8 Tregs along with CD8 suppressor cells in MM. A total of 91 monoclonal gammopathy patients were screened for CD8 Tregs and suppressor cells in peripheral blood (PB) and bone marrow (BM). For comparison, 21 healthy volunteers (HVs) PB were also analyzed. Clinical diseases of the patients were represented as monoclonal gammopathy of undetermined significance (MGUS)-14% (13/91), untreated MM- 60% (55/91) and relapsed MM (Rel MM)-26% (23/91). A simple three-color flow cytometric analysis was used to phenotype the CD8 Tregs and suppressor cells. CD8 Tregs were determined as CD8+FoxP3+ (Frisullo et al, 2010, Hum Immunol) and CD8 suppressor cells were identified by negative expression of co-stimulatory molecule CD28 (CD8+CD28-). All the results were summarized as percentage median and range. Our results showed strong significant increase in CD8 Tregs and suppressor cells in PB of patient cohort compared to HVs [CD8 Tregs- 0.39% (0.03%–2.41%) vs. 0.18% (0.04%–0.61%); P=0.002 and CD8 suppressor cells- 69.42% (10.99%–95.91%) vs. 27.58% (9.08%–70.52%); P<0.0001]. Additionally, strong significant difference was also noticed between HVs vs. MGUS, MM, and Rel MM for PB CD8 Tregs and suppressor cells; the data with statistical results has been depicted in table 1. More or less similar frequencies of PB CD8 Tregs were found between MGUS vs. MM vs. Rel MM. In term of PB CD8 suppressor cells Rel MM had insignificant increase compared to MGUS and MM. Surprisingly, MM cohort had insignificantly decreased frequency of CD8 suppressor cells compared to MGUS. As similarly to PB, BM CD8 Tregs and suppressor cells did not show any statistical significance in between patient groups. The BM CD8 Tregs frequency was observed respectively as: MGUS-0.32% (0.08%–1.04%), MM-0.44% (0.06%–1.82%) and Rel MM-0.27% (0.11%–1.16%). Respective BM CD8 suppressor cells frequency was noticed as: MGUS-63.32% (24.83%–94.43%), MM-62.15% (6.19%–93.45%) and Rel MM-67.29% (18.55%–87.61%). Subgroup of patients with < 95% of abnormal plasma cells [ANPCs- 59.21% (13.35%–91.83%)] had significantly decreased level of PB CD8 suppressor cells compared to counter group [≥95% of ANPCs- 74.23% (10.99%–95.91%)]; P=0.046. In line with PB, BM CD8 suppressor cells also showed significantly reduced frequency in <95% of ANPCs cohort [56.99% (16.48%–94.43%)] compared to ≥ 95% ANPCs cohort [69.61% (6.19%–93.45%)]; P=0.013. No relevant association was noticed between CD8 Tregs and suppressor cells with other clinical features including β2M, albumin, creatinine, lactate dehydrogenase, hemoglobulin and monoclonal protein level. In conclusion, our results disclose that as similar to CD4 Tregs, CD8 Tregs are also deregulated in MM compared to HVs; this is also in line with CD8 suppressor cells. This data suggest that in MM immune dysfunction is also enhanced by the shift in favor of CD8 immune regulatory and suppressor cells. This study was supported by the respective grants-MSM0021622434, LC06027, IGA NS10406, IGA NS10408, and GACR P304/10/1395. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Urinary abnormalities following karate (kumite) competitions.

Sir, Recently, our group conducted a supplementary study on urinary abnormalities following karate (kumite) competitions in professional female athletes. In a previous issue of NDT Plus [1], our group presented data showing significantly increased proteinuria of a mixed type (albumin and beta2-microglobulin or B2M) with prominence of albumin, as a glomerular urine protein, in 18 professional (18–21 years) male karatekas. This recent study has focused on 17 healthy (16–25 years) female karatekas, who volunteered to participate in the study, in order to detect karate-induced urinary abnormalities. The competitions were held with similar characteristics of three rounds of 1.5-min duration which had been interspaced with a 10-min resting interval. Urine samples were collected just before the match and 24 h thereafter to measure total urinary protein, albumin, B2M, sodium, potassium and calcium levels. All values were compared with pre-exercise 24-h urine values. There was significantly increased mixed-type proteinuria (total protein of 190.01 ± 92.54 vs. 68.6 ± 45.51 mg/day, P = 0.001; albumin of 30.07 ± 28.81 vs. 3.71 ± 2.3 mg/day, P = 0.001; and B2M of 0.0702 ± 0.0135 vs. 0.015 ± 0.01 mg/day, P < 0.01) compared with basal levels. Furthermore, similar to previous study, daily urinary electrolytes (sodium 121.65 ± 20.40 vs. 118.21 ± 21.25, P = 0.825; potassium 41.53 ± 10.80 vs. 43.59 ± 11.86, P = 0.426; and calcium 190.88 ± 80.44 vs. 182.76 ± 87.33, P = 0.755) excretions did not change significantly following the competitions. Urinary glucose in both pre- and post-exercise urine samples was not found. Based on the aforementioned data, it seems that karate (kumite) competition in both genders does not have significant influence on some urinary electrolyte and substance excretions. This finding may be attributed to appropriate tubular transport, short duration of competitions, adequate hydration of athletes and efficacious regulatory mechanisms in order to maintain normal blood levels [2]. However, because of the intensity of this exercise which results in sympathetic stimulation, the glomerular permeability and protein excretion may be changed [3,4]. We recommend further researches on karate-induced urinary abnormalities. Conflict of interest statement. None declared.

The Impact of β2-Microglobulin Clearance on the Risk Factors of Cardiovascular Disease in Hemodialysis Patients

beta2-Microglobulin (beta2M) is an independent predictor of outcome for hemodialysis (HD) patients and a representative substance of middle molecules. We tested the relationship among serum beta2M levels and cardiovascular disease (CVD) risk factors in HD patients. A total of 132 HD patients were divided according to the dialysis membrane used [property; cellulose and synthetic or beta2M clearance; low filtration (LF), middle filtration (MF), and high filtration (HF)]. There was no significant difference in CVD risk factors between cellulose and synthetic groups. On the other hand, serum beta2M, highly-sensitive C-reactive protein (hCRP), troponin-T (TnT), and myeloperoxidase (MPO) levels of LF were significantly higher and those of prealbumin (PA) were lower than the MF and HF. Serum beta2M level was positively correlated with hCRP, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), MPO, TnT, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and inversely correlated with PA and ankle-brachial index (ABI). There was a significant correlation between serum beta2M levels and various CVD risk factors in HD. Cardiovascular disease risk factors in HD patients were dependent on the beta2M clearance but not membrane property.

Predicting 10+ year survivors in total therapy 1 (TT1) for multiple myeloma (MM).

8138 Background: The importance of marked tumor regression for long-term survival in MM is well appreciated. With a median follow-up in TT1 for 231 newly diagnosed patients with MM of 16 years, median overall survival (OS) and event-free survival (EFS) durations are 5.7 and 2.6 years, with 47 patients remaining alive and 25 event-free including 12 of 87 who had attained CR. The purpose of this investigation was to determine the baseline features that were independently linked to 10+ year OS and EFS, in the context of responsiveness (partial response, PR; complete response, CR) in the first year of therapy. Methods: Baseline and response levels were compared between ≤10yr and >10yr survivors and logistic regression applied to identify the critical features linked to >10yr OS and >10yr EFS status. Results: Among traditional baseline prognostic variables distinguishing >10yr survivors (lower frequencies of: CA; elevated levels of B2M, LDH and CRP; decreased hemoglobin; higher proportions of patients attaining CR and PR within 1yr), multivariate analysis identified PR as an independent favorable variable (OR = 3.32, p = 0.01) while adverse implications were observed for CA-13 status (OR = 0.08, p = 0.01), LDH ≥190U/L (OR = 0.39, p = 0.04) and CRP ≥4mg/L (OR = 0.48, p = 0.02). When examined by immunoglobulin isotypes (IgG, IgA, only light-chain), PR remained favorable for IgG (n = 130) along with adverse implications of any CA and of female gender; while CRP was the only variable surviving the model for IgA (n = 41); and no one variable identified >10yr survivorship in the 37 patients with only light chain disease. >10yr EFS was positively linked to CR (OR = 2.56, p = 0.02) and negatively to CRP > = 4mg/L (OR = 0.31, p = 0.01) Conclusions: Long-term >10yr EFS required CR and >10yr OS PR to have been achieved within the first year of therapy; with CRP elevation affecting both endpoints adversely, while LDH elevation and CA-13 status at presentation were additional negative features for >10yr OS. No significant financial relationships to disclose.

Circulating Ki-67 index in plasma as a biomarker and prognostic indicator in chronic lymphocytic leukemia

Ki-67 is a nuclear antigen that is expressed in all stages of the cell cycle, except G 0, and is widely used as a marker of cellular proliferation in human tumors. We recently showed that elevated levels of Ki-67 circulating in plasma (cKi-67) are associated with shorter survival in patients with acute lymphoblastic leukemia. The current study included 194 patients with CLL and 96 healthy control subjects. cKi-67 levels in plasma were determined using an electrochemiluminescent immunoassay. We normalized the cKi-67 level to the absolute number of lymphocytes in the patient's peripheral blood to establish the plasma cKi-67 index. The cKi-67 index showed significant correlation with lymph node involvement and Rai stage ( P = 0.05). Higher cKi-67 index values were significantly associated with shorter survival. Multivariate Cox proportional hazards regression analysis demonstrated that the association of the cKi-67 index with shorter survival was independent of IgV H mutation status. In a multivariate model incorporating the cKi-67 index with B2M and IgV H , only cKi-67 index and B2M levels remained as independent predictors of survival. The results of this study suggest that the plasma cKi-67 index, along with B2M level, is a strong predictor of clinical behavior in CLL.

Electrophoretic analysis of various urinary proteins in Japanese patients with Dent disease

Patients with Dent disease excrete high amounts of urinary α1-microglobulin (α1-m), β2-microglobulin (β2-m), and retinol-binding protein (RBP). We found that the levels of α1-m, β2-m, and RBP in patients with Dent disease were higher than those in healthy subjects by 20 times, 130 times, and 200 times, respectively. These results confirmed the results of previous studies. The cellulose acetate membrane electrophoresis (CAE) showed that the characteristics of the urinary protein fraction in patients with Dent disease included appearance of pre-albumin, low albumin value, equal α1-globulin (α1-G) and α2-globulin (α2-G) values, disappearance of β-globulin (β-G), and appearance of slow α2-G (RBP) and slow β-G (β2-m). Urinary albumin (ALB), α1-m, β2-m, and RBP of patients with Dent disease 1 (mutation of CLCN 5) and Dent disease 2 (mutation of OCRL 1) and of healthy subjects were identified using western blot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Six bands from 84 kDa to 130 kDa were detected in addition to a main band of ALB (67 kDa). Seven bands from 51 kDa to 92 kDa were detected in addition to a main band of α1-m (34 kDa). A 17-kDa band was detected in addition to a main band of β2-m (14 kDa). RBP was detected only in the main band (17 kDa). The ALB and α1-m bands, which were in the high molecular weight range in addition to the main band, were densely stained in Dent disease 2.

Prognostic Value of Angiogenesis Assessment in Chronic Lymphocytic Leukemia: More Data Needed.

Abstract 4396 IntroductionThe association between angiogenesis and cancer progression in solid tumors has been documented, but its significance in chronic lymphocytic leukemia (CLL) has not been completely evaluated. Vascular endothelial growth factor /VEGF/ is the major pro-angiogenic factor in humans and his transduction pathway may be very active in CLL cells contributes to their enhanced survival. In this context VEGF can be additional tool for predicting the clinical course in early CLL. Patients and MethodsTo predict the risk of disease progression, we analyzed serum levels VEGF (sVEGF) using ELISA tehnique in 33 Binet stage A de novo CLL patients. In addition, We analyzed microvessel density (MVD) of the same patients using immunohistochemical staining with CD34 and vWf. Finally, we explored wheather changes of circulating VEGF concentrations and MVD reflected clinico-biological features of CLL (peripheral blood lymphocytosis (PBL), bone marrow (BM) histology, beta-2 microglobulin (β2m) level, lactat dehidrogenase (LDH) level) and kariotype abnormalities. ResultsThe VEGF serum levels (sVEGF) was not significantly elevated (p=0.31) in CLL patients (mean: 70.9 pg/mL; range: 16–483) compared with and age- and sex-matched healthy controls (mean: 44.7 pg/mL; range: 22–69.2). sVEGF level positively correlated with elevated LDH level (p=0.008), but no correlation with other clinico-biological features was found.Bone marrow MVD was significantly higher (p<0.0001) in CLL patients (mean: 35.91 vessels/field ± 15.71) compared to controls (mean:8.27 vessels/field ±6.19). Also, there was a significant difference between MVD counts according to the antibody used. MVD was higher using CD34 vs vWF (CD34, 35.91 vessels/field ±15.7, vs vWF, 8.15 vessels/field ±4.65, p<0.0001). Bone marrow MVD detected by CD34 was significantlyhigher in patinents with CD38 expression more than 30% (p=0.006). However, no significant MVD differences were detected between CLL subgroups with regard to clinical course, pattern of marrow infiltration, Rai stage and FISH abnormalities.In univariate analysis sVEGF (p=0.018), Rai substages (p=0.027), PBL (p=0,030), β2m level (p=0.008) and diffuse bone marrow infiltration (p=0.006) were significantly associated with increased risk of disease progression. But, in multivariate analysis only sVEGF (p=0.002) and β2m level (p=0.008) retained their prognostic significance. ConclusionsSerum VEGF level although not increased in comparasion with healthy controls, may improve the assessment of individual prognosis of patients with early CLL. Assessment of real clinical significance of bone marrow angiogenesis in CLL is required. Disclosures:No relevant conflicts of interest to declare.

Prognostic Significance of Beta-2 Microglobulin (B2M) in Adult Acute Lymphoblastic Leukemia (ALL).

Abstract 4679▪▪This icon denotes an abstract that is clinically relevant.Beta-2 microglobulin (B2M) is 12-kDa light chain protein that binds to the alpha chain of class 1 MHC molecules, dissociated into extracellular fluids after degradation of HLA. Elevated levels of serum B2M have historically been associated with a poor prognosis in several lymphoproliferative disorders. The prognostic relevance of B2M in myeloid disorders such as acute myelogenous leukemia and myelodysplastic syndrome has also been noted, in some cases providing further refinement of existing prognostic models. The significance of pretreatment serum B2M levels has not been extensively studied in adult ALL. In our historical experience with the conventional VAD (vincristine, doxorubicin, dexamethasone) regimen, elevated levels of B2M were associated with a lower complete remission (CR) rate, worse survival (OS), and higher propensity for development of CNS leukemia, after adjusting for renal dysfunction and older age [Kantarjian, Am J Med 93:599, 1992]. The analysis was somewhat limited by the strong negative impact of the mature B-cell ALL subset were the significantly high levels of B2M were associated with poor prognosis in the era prior to intensive multi-agent chemotherapy regimens. We assessed the associations of pretreatment B2M levels with outcome in 320 adolescents and adults with de novo ALL (Burkitt leukemia/lymphoma, Philadelphia chromosome positive ALL, and lymphoblastic lymphoma subtypes were excluded from the analysis) treated with the more intensive hyper-CVAD regimen (fractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone alternating with methotrexate and cytarabine) and its variants (modified hyper-CVAD with or without anthracycline intensification and with or without rituximab). Median serum B2M level was 3 mg/L (range 0-14.7). As observed previously, the CR rate was lower for patients (pts) with elevated levels of B2M, an effect which persisted after excluding the elderly subset and accounting for renal dysfunction (93% versus 98%, p=.02). The 3-yr rates for CRD and OS were also worse if B2M was elevated above the median, 55% vs 70% (p=0.09) and 60% vs 70% (p=.01), respectively. These findings confirm the prior observations in a larger more homogenous population of de novo ALL treated with intensive chemotherapy. A limited multivariate analysis identified the independent prognostic influence of B2M on survival. Additional analyses will be conducted to determine how these observations can be best incorporated into clinical practice. Disclosures:No relevant conflicts of interest to declare.

The Significance of Elevated Beta 2-Microglobulin (b2-m) in B-CLL: Evidence of in Vitro b2-m Secretion Following Activation of B-CLL Cells.

Abstract 4380Beta 2-microglobulin (b2-m) is an 11-kDa protein recognized as the light-chain component of the MHC-I molecule. It is produced by nucleated cells membranes and is detectable in the serum and other body fluids. The link between the b2-m/ MHC-1 molecules and membrane structure has been associated with lymphocyte activation. Serum b2-m is elevated in a variety of disorders and has a prognostic value in lymphoprolifera! tive diseases such as multiple myeloma and CLL. High b2-m in CLL, has been found to correlate mainly with the advanced stage of the disease and high lymphocyte count and is routinely used as a bad prognostic factor (Keating, 1995, Montillo, 2005). Previous studies from our Institute have shown significantly higher level of b2-m in CLL/PL and PLL and in cases associated with autoimmune disorders close to levels found in active myeloma (Shvidel, 1996, Duek, 2006). We suggested that b2-m levels are increased during B-cell activation which not necessarily correlates with aggravation of CLL. In an attempt to further evaluate the correlation of bm-2 and B-cells activation we stimulated B-CLL cells in vitro with pokeweed mitogen (PWM) and compared the results with CLL controls after 3 and 7 days of culture. Level of b2-m in supernatants was tested using a microparticle enzyme immunoassay. B-CLL cells from 8 patients (4 in stage Binet A, 3 B and 1 C) were studied. Except for stage A, the serum b2-m was mildly increased. The results showed a significant in vitro secretion of b2-m after three and seven days in supernatants of PWM-activated cells compared to unstimulated controls: mean values after 3 days were: 188 ng/ml (range 96-279) in activated B-cells versus 57ng/ml (25-103) in controls; and after 7 days 599 ng/ml (401-914) versus 164 (67-267) in controls. Interestingly, CD8+ T-cells from a T-large granular leukemia patient stimulated with PHA do not secrete significant amount of b2-m in vitro (156 ng/ml versus 87 at day 7). Our results demonstrate that activation of B-CLL cells in vitro induced a marked secretion of b2-m. Thus, the significance of b2-m in CLL may correlate with degree of activation of B-CLL cells. This hypothesis is supported by the increase of CD38 (an activation marker) in CLL cases with high b2-m serum level. Moreover, the results of our study suggest that progression of disease may be the results of in-vivo activation of the cells and lends support to previous studies showing that CLL cells are G0 cells with a potential to progress in the cell cycle after stimulation. Disclosures:No relevant conflicts of interest to declare.

Plasma Circulating Ki-67 Index as a Biomarker and Prognostic Indicator in Patients with Chronic Lymphocytic Leukemia.

Abstract 1261Poster Board I-283Ki-67 is a nuclear antigen that is expressed in all stages of the cell cycle except G0 and is widely used as a marker of cellular proliferation in human tumors. We recently demonstrated that levels of plasma circulating Ki-67 (cKi-67) are significantly higher in patients with newly diagnosed acute lymphoblastic leukemia (ALL) than in healthy control subjects, and that elevated levels of cKi-67 are associated with a shorter survival in ALL patients. Here we examined the associations of cKi-67 levels with laboratory and clinical variables in patients with chronic lymphocytic leukemia (CLL). The study included 194 patients with CLL and 96 healthy control subjects. The cKi-67 levels in plasma were determined using electro-chemiluminescence-based immunoassay using the Mesoscale Discovery platform. Since usually Ki-67 is used as an index of tumor cell proliferation, we took into account the lymphocyte count of the CLL patients in peripheral blood and normalized the levels of the cKi67 to the absolute number of lymphocytes in the peripheral blood establishing plasma cKI-67 index (cKi-67 level ng/1000 circulating lymphocytes/μL plasma). Median (range) levels of absolute cKi-67 were significantly higher in patients with CLL than in control subjects (914.65 [102.0-4975.12] ng/mL vs 353 [35.76-2830.65] ng/mL; P<0.0001). However, absolute levels did not correlate with clinical or other laboratory variables (white cell count, hemoglobin, platelets, beta-2 microglobulin, or Rai stage, performance status). In contrast, the cKI-67 index correlated significantly with bone marrow involvement (p<0.001), number of lymph node sites involved (p<0.001), and Rai stage (p=0.05), but not with IgVH mutation (P=0.62) or performance status (p=0.71) The cK-I67 index was significantly associated with survival when used as either as a continuous variable (P=0.002) or as a dichotomous variable (P=0.005). Multivariate Cox proportional hazards analysis incorporating cKi67 index with IgVH mutation status and B2M, demonstrated that only cKi-67 and B2M were independent predictors of survival.This data shows that there variability in proliferation between patients with CLL and those patients high relative proliferation (index) have more aggressive disease. Furthermore, plasma cKi-67 index and B2M levels are strong predictors of clinical behavior in CLL. DisclosuresNo relevant conflicts of interest to declare.

Maintenance Rituximab Is Associated with Improved Progression Free and Overall Survival in Waldenstrom's Macroglobulinemia.

Abstract 3750Poster Board III-686Rituximab is an important mainstay of therapy in patients with Waldenstrom's macroglobulinemia, though the benefit of maintenance rituximab has not been established. As such, we examined the outcome of patients followed at our Institution who responded to a rituximab based therapy, and assessed the impact of maintenance rituximab. Two hundred forty eight patients were included in this analysis, of whom 86 (35%) received maintenance rituximab following response. No difference in baseline age, serum IgM, IgA, IgG, BM disease involvement, complete blood counts, B2M levels, as well as categorical response rates (CR, VGPR, PR and MR) following induction therapy were observed between cohorts. 181 (73%) patients were previously untreated, with a similar proportion in both cohorts. Induction therapy for all patients included rituximab alone (n=79), or in combination with bortezomib (n=40); cyclophosphamide (n=44); immunomodulatory agent (n=31); or a nucleoside analogue (n=54) containing regimen. The median number of rituximab infusions given as induction was 6 for both cohorts, while the median number of rituximab infusions given as maintenance was 8. Maintenance rituximab was administered as 1 infusion (at 375 mg/m2) every 3 months for 63 (73%) patients, and as 4 weekly infusions (at 375 mg/m2) every 6 months for 23 (27%) patients, with a median of 2 years of treatment. Both progression free (56.3 vs. 28.6 months; p=0.0001) and overall survival (>120 vs. 116 months; p=0.0095) were longer in those patients who received maintenance rituximab (Figs. A, B). Improved progression free survival was evident despite previous untreated or treated status, induction with rituximab alone or in combination therapy, as well as induction with a cyclophosphamide, nucleoside analogue or bortezomib containing regimen (p=0.0001). Among patients receiving maintenance rituximab, progression free survival was 53.3 versus 61.3 months (p=0.4931) for those patients receiving one infusion of rituximab every three months compared to those receiving 4 weekly infusions every 6 months, respectively. Best response serum IgM was 598 vs. 1380 mg/dL (p<0.0001), and hematocrit was 40.7% vs. 38.6% (p=0.001) in patients who received maintenance versus no maintenance, respectively. Categorical response improvement was also observed in 33/86 (38.4%) patients following maintenance therapy. Among patients receiving maintenance therapy, median serum IgG (351 vs. 461 mg/dL; p=0.0008) and IgA (23 vs. 33 mg/dL; p=0.09) levels were lower, and an increased number of infectious events during the course of follow-up were observed (p=0.008) which were mainly sinopulmonary though ≤grade 2. The results support the use of maintenance rituximab in WM patients who respond to induction with a rituximab containing regimen, and highlight the need for further studies aimed at clarifying the ideal schedule and duration of maintenance rituximab therapy in WM. [Display omitted] Disclosures:Treon:Genentech BioOncology Inc.: Consultancy, Honoraria, Research Funding; Biogen IDEC Inc.: Consultancy, Honoraria, Research Funding.

Serum FLC Levels at Presentation Have Independent Prognostic Significance in CLL and Levels Above 50mg/L Identify Patients with Progressive Disease.

Abstract 2355Poster Board II-332An abnormal serum FLC ratio at presentation has been shown to be prognostic in chronic lymphocytic leukaemia (Pratt et al, 2009). Here we further analyse a retrospective cohort of 259 CLL patients (Stage: A, 209; B 23, C, 21; Male: Female ratio 1.6:1, mean age 75: Range 29-98). The levels of FLC and B2M were assessed using nephelometric immunoassays (The Binding Site, Birmingham UK) on the Siemens Dade Behring BN‘II analyser. Previously recorded measurements for biological and clinical markers (age, sex, CD38, Zap70, and VH mutational status) were used to produce Kaplan Meier Survival Curves and in Cox Regression analysis. We have identified that a cut-off above 50mg/L (in the context of an abnormal ratio to exclude renal effects) identifies a cohort of patients with progressive disease and a significantly poorer outcome. A total of 38 out of the 259 patients had serum FLC > 50mg/L. CLL patients with serum FLC >50mg/L had the following characteristics Stage A/B/C (26/5/5 + 2 unknown), Mutated vs Unmutated (19 vs 13, 6 unknown), Zap70 pos vs neg (19 vs 16, 3 unknown), CD38 pos vs neg (13 vs 23, 2 unknown) and 32 out of the 38 patients have progressed to treatment.Median time to first treatment for the CLL cohort with >50mg/L serum FLC was 83 months compared to 241 months for the CLL patients with normal FLC (p=9.8×10−6). Median overall survival was also significantly shorter for patients with an abnormal ratio and >50mg/L serum FLC (p=7.6 × 10−7). Cox regression analysis on the above population gave stage (Hazard ratio 3.9 p=<0.001), Zap 70 (Hazard ratio 1.9, p=0.001) and abnormal ratio with production above 50mg/L (Hazard ratio 1.9 p=0.009) as the only independent prognostic variables.Importantly >50mg/L FLC production is independent of both Zap70 and Vh mutational status as an indicator of time to first treatment. This study shows that in an unselected population of CLL patients serum FLC >50mg/L can independently identify a group of CLL patients with progressive disease and a poorer outlook. Disclosures:Harding:The Binding Site Group Ltd: Employment. Bradwell:The Binding Site Group Ltd:. Mead:Binding Site UK Ltd: Employment.