IgVH Mutational Status Research Articles

Pharmacological targeting of eIF4E in primary CLL lymphocytes

Pharmacological targeting of eIF4E in primary CLL lymphocytes

Autoimmune disorders in patients with B-cell chronic lymphocytic leukemia.

7103 Background: B-cell chronic lymphocytic leukemia (CLL) results from the accumulation and proliferation of malignant B cells with a predisposition for autoreactivity and autoimmune disease, particularly hematologic disorders such as autoimmune hemolytic anemia (AIHA) and immune thrombocytopenic purpura (ITP). We sought to define the prevalence of autoimmune conditions and associated prognostic factors in our CLL cohort. Methods: We retrospectively reviewed the electronic medical records (EMR) of CLL patients at Duke University Medical Center (DUMC) and Durham VA Medical Center (DVAMC) for known diagnoses of autoimmune conditions, or clinical descriptions matching those disorders. Laboratory data confirming the diagnoses (e.g., Coombs test for AIHA) were verified directly in our EMR. IGVH mutation status, CD38 and ZAP-70 expression were previously determined. Results: We found 92 CLL patients with autoimmune disorders (21.4% prevalence), with higher numbers of both hematologic (58, 11.1%) and non-hematologic(42, 8.1%) conditions than previously published data [Barcellini et al. Haematologica (2006) 91:1689], although one series did show 9.7% of CLL patients with hematologic phenomena [Hamblin et al. J Clin Path (1986) 39:713-6]. CLL patients with AIHA and/or ITP had significantly shorter treatment-free survival [2.1 years (1-3.3)] and overall survival [11.9 years (9.6-15.8)] than those without [6.2 years (5.2-7.3) and 18.0 years (14.0-22.6), respectively]. 52% of CLL patients with autoimmunity had mutated IGVH (41/79 known results), 18% were CD38 positive (16/89) and 49% were ZAP-70 positive(39/80). In CLL patients without autoimmune disorders, the prevalence of mutated IGVH, and CD38 and ZAP-70 positivity were similar at 60% (219/364), 25% (99/395), and 47% (176/377), respectively. Conclusions: There is a higher prevalence of both hematologic and non-hematologic autoimmune disorders in our CLL cohort than previously reported. CLL patients with hematologic autoimmune disorders had shorter time to treatment and survival, compared to those without. There was no difference in frequency of mutated IGVH, CD38 or ZAP-70 expression between those patients with autoimmune conditions and those without.

Abstract 932: Predictive markers of the in vitro anticancer effect of the pan class I PI3K inhibitor BKM120 in primary B-CLL lymphocytes.

Abstract B-chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+ B-lymphocytes that are long-lived in-vivo but die quickly by apoptosis when cultured in-vitro. The phosphatidylinositol-3 kinase (PI3K) cascade which is a critical component of survival signaling has increased activity in CLL lymphocytes as compared to normal B lymphocytes. There are three classes of PI3Ks of which class I is the most clearly implicated in human cancer. The PI3K-δ inhibitor, CAL-101, promotes apoptosis and abrogated protection from spontaneous apoptosis induced by CD40 in primary CLL lymphocytes in-vitro. Thus the PI3K pathway appears to play a critical role in B-CLL cell survival. BKM120 is a pan class I PI3K inhibitor developed by Novartis. Phase I trials demonstrated that plasma concentrations (Cmax) of 5 μM can be obtained. In view of the critical role of PI3K in CLL homeostasis, the activity of BKM120 was examined in CLL lymphocytes in-vitro. BKM120 was cytotoxic in the 3 CLL cell lines and in 78% of the primary B-CLL lymphocytes samples isolated from 65 B-CLL patients, including patients with del11 and del17. Interestingly, 60% of the B-CLL samples tested in our study have an IC50 below the Cmax. Furthermore, BKM120 cytotoxicity correlated with the basal expression of proteins involved in the PI3K/Akt pathway (Akt, rictor, raptor, p70S6K and 4E-BP1) but not with PTEN, mTor, IgVH or CD38 expression in these primary B-CLL lymphocytes. Further analysis of these different markers demonstrated that only patients very sensitive to BKM120 (IC50 ≤3 μM) expressed low basal levels of mTor, raptor and p70S6K simultaneously. Inhibition of PI3K was demonstrated by detection of phosphorylated Akt (S473) after BKM120 treatment in the cell lines. Molecular biomarkers such as p70S6K and 4E-BP1 have been used as indicators of PI3K pathway inhibition in-vivo. We also have demonstrated that BKM120 decreased the phosphorylation status of 4E-BP1 and p70S6K in the primary B-CLL lymphocytes tested. As detected by AnnexinV/propidium iodine staining and analysis of caspase-3 cleavage, BKM120 induced apoptosis in primary B-CLL cells culture in the presence and absence of stromal cell support. Taken together, our results demonstrated that the class I PI3K inhibitor BKM120 may be useful as a single agent in CLL patients independently of their IgVh mutational status, CD38 expression or genomic deletions (del11 and del17). Furthermore, a combination of different biomarkers (expression of mTor, raptor, and p70S6K) may predict the response to BKM120 treatment. Also, BKM120 abolished the protection against apoptosis and drug resistance conferred by the microenvironment to the primary B-CLL lymphocytes in-vitro. Our preclinical study demonstrated that BKM120, a molecule with acceptable clinical toxicity, may be useful in the treatment for CLL. Citation Format: Lilian Amrein, May Shawi, Jeremy Grenier, Raquel Aloys, Lawrence Panasci. Predictive markers of the in vitro anticancer effect of the pan class I PI3K inhibitor BKM120 in primary B-CLL lymphocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 932. doi:10.1158/1538-7445.AM2013-932

Abstract 2852: Obinutuzumab (GA101) efficacy in chronic lymphocytic leukemia in vitro is not diminished in high risk patients.

Abstract Introduction : we and others have already demonstrated the superiority of obinutuzumab (GA101) over rituximab (RTX) in in vitro depletion assays against chronic lymphocytic leukemia (CLL) cells. It has been recently suggested that complex karyotype (3 or more chromosomal abnormalities) and recurrent somatic mutations of the TP53, NOTCH1 and SF3B1 genes, influenced treatment-free and overall survivals in CLL patients. We address the question whether these factors may also influence the efficacy of anti-CD20 directed immunotherapy. Aim: To assess in vitro pre-clinical activity of RTX and GA101 against CLL cells in freshly isolated PBMCs, and correlate depletion efficacy with modern and classical (FISH, IgVH mutational status, age, gender, Binet stage, b2-microglobulin, bulky adenopathies>5cm) prognostic parameters in 95 CLL patients. Moreover, CD20 antigen density was assessed in 24 patients. Methods: PBMCs were isolated from blood samples by Ficoll gradient centrifugation. Antibody-mediated B cell depletions were determined by enumerating trypan blue negative, flow cytometrically CD19-positive B lymphocytes after treatment with 10 μg/ml of anti-CD20 antibodies. The Quantibrite® kit was used to determine CD20 density, measured as quantity of antibodies bound per cell (CD20-ABC). The Mann-Whitney test was used to compare median depletion according to relevant clinical and biological data. Results: patients characteristics were as follows: 69% males, median age 66y (36% >70y), advanced Binet stage B/C in 39% (31.7% had NCI2008 criteria for treatment), 22.6% had lymph nodes>5cm (56% had b2-microglobulin>3.5mg/l). Molecular and cytogenetics studies were as follows: unmutated IgVH in 42.7%, del13q/tri12/del11q/del17p=25.6%/22%/20%/5.8% respectively, complex karyotype (CK) was found in 20.8% of cases, and somatic mutation of TP53/NOTCH1/SF3B1 in 11.1%/12.6/4.8% of patients. The median B-CLL depletion in freshly isolated PBMC fraction due to treatment with were 61% for GA101 vs 21.5% for RTX (n=95, p<0.001). GA101 had higher activity than RTX in 83% of patients, yielding B-cell depletion>50% in 64% of cases. As shown in Figure 1, in all categories of patients, GA101 was superior to RTX. This was particularly noticed in the patients with TP53 deletion and/or mutation (n=7). Median CD20-ABC was 8100 in 24 patients, who were categorized as high or low CD20 expression based on that median. Low CD20-ABC patients had significantly less antibody-induced B-cell depletion, 12.8% (vs 25%, p=0.37) for RTX, and 42.5% (vs 67.5%, p=0.048) for GA101. CD20-ABC was not found to be correlated with clinicobiological parameters described above. Conclusions: according to modern prognostic parameters, GA101 confirms its higher efficacy than RTX (three-fold), and deserves further combination with chemotherapy or non-chemotherapy entities in CLL management. Citation Format: Loic Ysebaert, Emilie Laprévotte, Christian Klein, Jean-Jacques Fournié, Anne Quillet-Mary. Obinutuzumab (GA101) efficacy in chronic lymphocytic leukemia in vitro is not diminished in high risk patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2852. doi:10.1158/1538-7445.AM2013-2852

Is Chronic Lymphocytic Leukemia Still Incurable?

AbstractAbstract 3929 IntroductionUp until now, Chronic Lymphocytic Leukemia (CLL) has been considered incurable except with an allogeneic stem cell transplant. In the last 10 years, evidence has demonstrated that chemo-immuotherapy with fludarabine, cyclophosphamide, and rituximab, (FCR) has significantly improved CR rates, overall survival (OS), and progression free survival 1. Long term follow up data for FCR at MDACC demonstrated that a significant proportion of patients (pts.) were free at 8 years2 raising the question of whether pts. are potentially cured.Definition of cure in a chronic disease such as CLL has not been addressed. To investigate this possibility we evaluated the outcome characteristics of 222 of the 300 patients (who commenced FCR more than 10 years ago) in our previously reported study of initial therapy with FCR in CLL2.Seventy eight (35%) pts were free of relapse and 127 (58%) were alive at 10 years (Fig. 1). Thirty three patients died in CR/PR of infection (5), second malignancies (8), Richter's transformation (8). MDS (9), and other causes (3). One hundred and sixty three pts. (73%) achieved CR, 22 pts. (10%) a nodular PR (nPR), 27 pts. (12%) a PR, and 10 (5%) failed treatment. The 10 year PFS correlates strongly with response, CR (41%), nPR+PR (15%) (Fig. 2). None of these patients were transplanted in remission.FISH analysis was not available at the time of this study. Conventional karyotyping demonstrated +12 (24 pts), del 11q (15), del 17p (4), other abnormalities (15), diploid (105), and in 59 patients the test was not done or had no metaphases. The worst outcomes were del 17p and del 11q each significantly inferior to diploid (+12) patients had the best time-to-treatment failure (TTF) with P<.09 compared to diploid.The 10-year TTF was significantly higher for Rai <3 versus 3 – 4 (P=.02), serum beta-2-microglobulin (β2M) value of ≤ 4 mg/L (p<.001), mutation status of IgVh (P<.001) and number of courses of FCR received. (Table 1) The causes for receiving <6 courses of FCR were persistent cytopenia (15), infections (6), resistance (5), Richter’s transformation and other malignancies (9), autoimmune hemolysis (5), and other causes and lost to follow-up (16).Table 110 yr TTF10 yr OSCharacteristicValuePts.CR (%)Χ2TTF (%)p-valueOS (%)p-valueRai Stage<3/3-4145/7778/50.0438/22.0264/50<.01Serum b2M<4/>4129/9583/58<.00144/22<.00174/40<.001IgVh MutationMut/Unmut.47/8283/77ns49/11<.00176/46<.01# Courses<6/657/16551/81<.00116/38.00270/31<.001Of the 77 patients who are still in remission at 10 years, two have relapsed and one developed Richter's transformation. Four of 21 patients checked had no residual disease in their blood at 10 years by 4-parameter flow cytometry. All other 10 yr. TTF patients are being contacted to provide blood for 4-parameter flow residual disease. ConclusionThe present data suggest that one-third of patients treated with chemoimmunotherapy are potentially cured of CLL. The characteristics most strongly associated with 10 yr. TTF were Rai stage, serum β2M level, mutation status, and tolerance of chemotherapy. Second malignancies and transformations are emerging as significantly impairing the likelihood of cure. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Epigenetic Profiling of Primary CLL Reveals Novel DNA Methylation-Based Clusters and Novel Mechanisms of Leukemogenesis

AbstractAbstract 3877 Introduction:Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world with nearly 15,000 new cases diagnosed every year in the USA. The characterization of CLL has resulted in the identification of important disease biomarkers: these include the recurrent genomic deletions del17p and del11q, genomic complexity, TP53 mutations, the expression level of ZAP70 and the mutational status of IgVH. While genomic and transcriptional profiling of CLL identified clinically and biologically relevant markers, there is still significant uncertainty about the pathobiology and the origin of CLL. It is increasingly clear that epigenetic deregulation plays an important role in the biology of all lymphomas/leukemias including CLL. Methods:We hypothesized that DNA methylation profiling would allow us to identify new, biologically significant CLL subtypes and yield greater insight into the biology of this disease. We therefore examined the DNA methylation of over 240 patients with CLL using the HELP assay and hybridization to high density custom microarray that reports on the methylation status of more than 250,000 CpGs corresponding to 20,401 genes. Gene expression profiling and SNP array-based copy number assessments and targeted gene resequencing were available on most of these cases. We performed unsupervised analysis on the most variable probesets (standard deviation > 1.3) using K-means consensus clustering. Results:The experimental approach reproducibly identified three robust CLL subtypes based on epigenetic profiles. To identify the genes that define these three subtypes we next performed unequal variance t-test of the CLL subtypes comparing them to Peripheral Blood CD19+ B cells as a normal control, and identified that clusters are defined by differential methylation of 3719, 6145 and 3349 genes (selected probes displayed changes in methylation of at least 30% and FDR corrected p-value < 0.05), The three clusters featured respectively i) aberrant methylation of MYC and WNT target genes, ii) aberrant methylation of NOTCH1 targets and iii) aberrant methylation of bcl6 and inflammatory cytokines. There was inverse correlation between gene expression and cytosine methylation, suggesting that DNA methylation had an impact on the transcriptional programming of these CLL cases. Strikingly the CLL MYC/WNT cluster displayed poorer prognosis as opposed to the CLL BCL6 cluster (HR=0.14 95% CI: 0.07–0.30). The CLL NOTCH1 cluster had an intermediate prognosis. It was also notable that all CLL patients exhibited deregulation of the B-cell receptor pathway as compared to normal CD19+ B-cells, consistent with the notion that this pathway plays a critical role in CLL pathogenesis. Finally, we divided the cohort into training and testing cohorts and used a machine learning BDVAL algorithm to identify DNA methylation outcome classifiers. This procedure identified a 40-probeset classifier that accurately predicted outcome (Area Under the ROC Curve of 0.77; performance was assessed with 10 fold cross-validation in a training set with 76 patients; validation on an independent set of 105 samples). Conclusion:This large epigenetic profiling study in CLL identifies aberrant epigenetic regulation as a core part of the pathobiology of CLL and identifies novel CLL clusters with distinct effects on survival. MYC-WNT pathway inhibitors are warranted for use in clinical trials for patients belonging to this aggressive epigenetically defined subtype. Disclosures:No relevant conflicts of interest to declare.

BOTH MD Anderson Cancer Center MODEL and German Score Work in Predicting Time to First Treatment in EARLY Chronic Lymphocytic Leukemia: Results of an External Validation Analysis

AbstractAbstract 3934In chronic lymphocytic leukemia (CLL) there has been a limited published work on the development of models predicting time to first treatment that incorporate traditional and novel prognostic factors. We wondered whether the nomogram recently proposed by M D Anderson Cancer Center (MDACC) investigators that includes clinical and newer prognostic variables (i.e., three or more involved lymph node sites, increased size of cervical lymph nodes, presence of 17p deletion or 11q deletion by FISH, increased serum LDH, and unmutated IGHV mutation status) could work when applied to a prospective community-based CLL patient cohort.We considered suitable for the purpose of this study 328 previously untreated patients with newly diagnosed asymptomatic Binet stage A CLL from different primary hematology centers registered on a prospective basis during the period 2006–2010 on an observational database of Gruppo Italiano Studio Linfomi (GISL).Using the formula proposed by Wierda et a total point score was calculated for each patient. A value ranging between 0 and 69.5 (median, 19.9) - therefore similar to total point scores reported by the MDACC group (median, 21.0; range, 0 – 87.4) – was found. As a continuous variable (i.e., measuring the risk of each point increase), total point score was associated with time to first treatment (HR 1.04; 95% CI 1.02–1.05; P < 0.0001). Receiver Operating Characteristic analysis identified a point score of 25 [area under curve (AUC) 0.64, sensitivity 61.5 specificity 72.1; P<0.0001)] as the best threshold capable of separating patients who needed therapy from patients who did not (HR, 3.27; 95% CI, 2,07–5.18; P<0.0001). The prognostic index category remained a predictor of time to first treatment also when analysis was limited to patients in Rai stage 0 (HR,4.05; 95% CI: 2.25–7.52; P<0.0001). Finally, the goodness of fit showed that the nomogram model had significant good fit at 2 years (r2=0.966; P=0.002).We next wondered whether the German score based on 7 prognostic variables (i.e., age, gender, b2-microglobulin, sTK, IGVH mutational status, ECOG performance status and hierachic type of FISH abnormalities) and originally designed for predicting overall survival could forecast time to first treatment in patients with early disease. Since sTK was not available a slightly modified version of German model based on 6 prognostic variables was used. The risk score calculated in each patient by summing single ratings of adverse factors, allowed the identification of two different risk categories scoring respectively 0–2 and 3–5 whose clinical outcomes were different with respect to time to first treatment (HR 2.19, 95% CI: 1.15–2.20; P=0.005).In conclusion, this analysis shows that both models proposed by MDACC investigators and German group provide interchangeable tool for assessing time to first treatment. Interestingly, in this validation analysis the pattern of distribution of patients into a low and high-risk category revealed a high degree of concordance between models (P<0.0001; Mann-Whitney test). Disclosures:Morabito:Celgene: Honoraria.

Overexpression and Aberrant Nuclear Translocation of NFAT2 in CLL

AbstractAbstract 3909Chronic Lymphocytic Leukemia (CLL) is a hematological malignancy of mature B cells and constitutes the most common leukemia in adults. It is characterized by a progressive accumulation of clonal B cells, which coexpress CD19, CD23 and CD5. The clinical course of CLL can be predicted by serveral prognostic markers like CD38, ZAP70 and cytogenetic abnormalities. While the treatment of CLL has significantly improved during recent years, it remains an essentially uncurable disease and the molecular events that lead to its development are still largely ellusive. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated by a network of several kinases. Several recent studies have demonstrated that Ca2+/NFAT signaling is involved in the pathogenesis of a wide array of different tumor types including pancreatic adenocarcinoma, breast cancer and Non Hodgkin′s lymphoma. In this study we investigated the significance of the Ca2+/NFAT signaling pathway in B-CLL.For this purpose, we analyzed CLL cell lines (MEC-1, JVM-3) as well as primary blood samples from patients with CLL (n=30). The analyzed patient population exhibited a representative distribution of age, sex, Binet stage, WBC count, cytogenetics and IGVH mutational status. PBMC were obtained by ficoll density gradient centrifugation and B cells were subsequently isolated using magnetic bead technology. NFAT2 expression and aberrant nuclear translocation was then assessed by Western Blotting and Immunofluorescence staining. In addition, NFAT2 mRNA levels were measured by qRT-PCR and its DNA binding capacity was assessed using an electrophoretic mobility shift assay (EMSA). Transcriptional activity of NFAT2 proteins in CLL cells was further analyzed by determining the expression of several well characterized NFAT target genes by qRT-PCR and Western Blotting.In our analysis, we detected a profound overexpression of NFAT2 mRNA as well as NFAT2 protein in all CLL samples. Using qRT-PCR we found that CD19+CD5+ CLL cells exhibited an at least three fold overexpression of NFAT2 as compared to CD19+ B cells isolated from healthy donors. In one case, NFAT2 expression in CLL cells was 200 times higher than in the corresponding control cell population. This profound overexpression of NFAT2 in CLL cells could be confirmed on the protein level using Western Blotting and Immunocytochemistry. We could further demonstrate that even under resting conditions significant amounts of NFAT2 protein had translocated to the nucleus in CLL cells, whereas virtually all NFAT2 was in the cytoplasm in healthy B cells. NFAT2 nuclear translocation could be inhibited using pretreatment with Cyclosporin A demonstrating that this process was still calcineurin-dependent in CLL cells. We could further show that nuclear NFAT2 in CLL cells was able to bind DNA but that its transcriptional activity with respect to several apoptosis and cell cycle regulating genes was severely compromised when compared with healthy CD19+CD5+ B cells.In summary, these results provide strong evidence that the Ca2+/NFAT signaling axis is con-stitutively activated in CD19+CD5+ CLL cells. Reduced expression of several apoptosis and cell cycle regulating proteins which are known target genes of NFAT2 potentially links deregulation of this signaling cascade to CLL progression. This is in line with our observation that genetic loss of NFAT2 can induce acceleration of CLL in TCL1 mouse model. Further investigation is warranted to decipher the therapeutic potential of modulating Ca2+/Calcineurin/NFAT signaling in CLL. Disclosures:No relevant conflicts of interest to declare.

High Expression of Lymphoid Enhancer-Binding Factor 1 (LEF1) Is Related to Ummutated Status of Igvh and Is an Unfavorable Prognostic Marker for Aggressive CLL Patients

Abstract 4573Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal CD5+ B lymphocytes. CLL is highly variable and heterogeneous in clinical courses, prognosis and outcomes, so efficient prognostic markers are essential to better identify different clinical courses of CLL. The prognostic influence of mutations in IGVH, NOTCH1, and other genes, as well as high expression of CD38 and ZAP70 has been identified in recent years. However, these prognostic markers are still not enough to accurately evaluate clinical courses and outcomes of CLL patients. The canonical Wnt pathway is widely involved in multiple physiological and pathological processions. As a member of the TCF/LEF family, lymphoid enhancer-binding factor-1 (LEF1) plays a critical role in Wnt pathway by directly interacting with β-catenin. LEF1 has a regulatory effect in cell proliferation, development and apoptosis of hematopoietic tissues, especially lymphoid lineages. Recent reports identified the potential participation of LEF1 in the pathogenesis of both B-cell and T-cell acute lymphoblastic leukemia. In CLL, it has been demonstrated that LEF1 is significantly overexpressed, and serves as a prosurvival factor for CLL cells.In the present study, we analyzed the relevance of LEF1 expression with clinical characteristics and outcomes in a cohort of 100 CLL patients. All the patients were previously untreated, or did not receive any treatment in the latest 6 months. The median age was 69 years. LEF1 expression was assessed using quantitative real-time PCR and β-actin mRNA served as an inner control gene. We found that LEF1 expression was significantly relevant with the somatic mutation status of IGVH (P<0.05). In patients with unmutated IGVH, LEF1 expression level was far higher than patients with mutated IGVH. We also found a correlation between LEF1 expression and ZAP70 protein expression. LEF1 mRNA expression level is higher among ZAP70-positive patients. However, this correlation is not statistically significant. No obvious association between LEF1 expression and CD38 was found. Patients were classified into high and low LEF1 subgroups according to a LEF1 media split. Then we analyzed LEF1 effect on overall survival (OS) in CLL patients in different clinical stages classified according to Binet or Rai staging system. Among patients in Binet C stage, high LEF1 expression predicted a shorter OS compared with low LEF1 patients (median, 120 months vs. undefined, P<0.05). For patients staging in Rai III-IV, patients with high LEF1 expression had a much shorter OS than low LEF1 patients (median, 108 months vs. undefined, P<0.05). As CLL patients generally share a relative longer term of survival, we concentrated mainly on the patients with a follow-up for more than 24 months for survival analysis. Low LEF1 patients, compared with high LEF1 patients, had a prolonged overall survival (undefined vs. 144 months; P=0.068). Although the correlation between LEF1 expression and overall survival did not get statistically significance yet, the correlation is quite obvious, indicating the prognostic potential of LEF1 for CLL patients.In summary, our data showed that LEF1 expression level is significantly correlated with IGVH mutation status. High LEF1 level is an unfavorable prognostic marker for CLL patients in aggressive stages and patients with a long clinical course. The results suggest that LEF1 may be involved in the pathological process of CLL and has the potential for serving as a prognostic marker of CLL. Disclosures:No relevant conflicts of interest to declare.

The Pro-Inflammatory IL23/IL23R/IL17 Axis Is Active in IL23R-Expressing Circulating CLL Cells in Patients with Poor Prognosis

AbstractAbstract 3889Inflammatory cytokines play a biological role in the pathogenesis of Chronic lymphocytic leukemia (CLL). IL23 is a pro-inflammatory cytokine involved in T-cell responses and in tissue remodeling. It has been shown that the IL23 receptor (IL23R) is up-regulated in primary acute lymphoblastic leukemia (ALL) cells, and that IL23 inhibits ALL cell growth. Nevertheless, the anti-tumor function of IL23 still remains controversial. The role of the IL23R/IL23 axis in CLL has not been investigated so far. Herein we evaluated the expression pattern of IL23R/IL23 axis and its correlation with progression free survival (PFS) in CLL patients.A total of 233 newly diagnosed Binet stage A CLL cases from Italian institutions (clinicaltrials.gov NCT00917540) were studied for IL23R expression by flow-cytometry (FC) (median percentage IL23R expression=22.7, range 1.2–91.1). The median follow-up was 23 months (range 1–47). PFS information was obtained in 203 patients. Using the median value of 23% of IL23R as threshold, 8/102 IL23Rneg and 23/101 IL23Rpos CLL cases progressed with therapy requirement. The 2-year PFS probability of IL23Rneg patients was 89.7% as compared to 80.7% of IL23Rpos cases [χ2 7.7, P=.006; HR=3.0, 95%CI (1.3–6.6)]. Cases were then stratified according to IL23R positivity [IL23Rneg (102 cases) versus IL23Rpos (101 cases)]. No significant difference in terms of CD38 and ZAP-70 positive cases was observed, however, the IGVH mutational status could distinguish the two groups: IGHV-mutated in 92 (78.6%) of IL23Rneg vs 70 (61.9%) IL23Rpos and IGHV-unmutated in 25 (21.4%) vs 43 (38.1%), p=.006]. FISH analysis showed that IL23Rneg and IL23Rpos cases carrying 13q14.3 were respectively 53 (51.4%) and 44 (42.7%), while the number of patients with trisomy 12 were 8 and 10 respectively in cases with low and high IL23R expression. Deletion of 11q was detected in 3.9% (4/103) of IL23Rneg and in 8.7% (9/103) of IL23Rpos cases. Only 3 cases with 17p deletion were seen in this cohort of early CLL patients and all belonged to the IL23Rpos group. Overall, no significant differences in the incidence of the major genetic lesions were observed between the two groups. Il23R expression still remained independently associated with PFS also in multivariate analysis.In situ expression analysis of IL23R and of its ligand IL23 was then performed by immunohistochemistry (IHC) in 16 CLL samples [10 lymph node (LN) and 6 bone marrow (BM) biopsies] collected on diagnosis and in 8 control biopsies (4 lymph nodes with reactive follicular hyperplasia and 4 normal BM biopsies). IL23R was variably expressed in CLL and significantly expressed in the neoplastic clones of 9 (6 lymph nodes and 3 BM biopsies) of the 16 cases tested; IL23R was diffusely present along the membrane and cytoplasm of neoplastic cells effacing the lymph node or BM architecture (Fig. 1, upper-left). In CLL cases with low IL23R expression, IL23R was detected in few scattered lymphoid cells intermingling with neoplastic lymphocytes (Fig. 1, upper-right). IL23 was also detected, with a variable staining intensity (Fig. 1, middle-left), paralleling in part that of IL23R. Double-marker analysis confirmed the concomitant expression of IL23 and IL23R in CLL neoplastic infiltrates highlighting the co-localization of the two markers (Fig.1 middle-right) and suggesting the possibility of an autocrine IL23/IL23R loop in CLL clones.We speculated that the microenvironment of CLL cases rich in IL23R and IL23 could be enriched in IL17-producing cells. The IHC expression of IL17 in CLL cases with low or high IL23R and IL23 expression showed that CLL cases rich in IL23Rpos cells, also characterized by high IL23 expression, displayed significantly higher numbers of IL17pos infiltrating cells (Fig. 1 bottom-left), as compared with CLL cases with no or low expression of IL23R or IL23 (Fig. 1 bottom-right).In conclusion, our study shows that high IL23R expression predicts a worse PFS. Furthermore, we linked this picture with, the in situ engendering of a clone-related microenvironment characterized by the preponderancy of pro-inflammatory signals such as those of the IL23/IL23R/IL17 axis, and its correlates in the peripheral blood (i.e. IL23R expression on circulating CLL cells), may endorse its strong prognostic significance. This analysis prompts further investigation into the specific function of the IL23/IL23R/IL17 axis and its targets in the context of CLL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Green Tea Extract EGCG Induces Apoptosis in CLL Cells and Overcomes the Supportive Effect of Primary Bone Marrow Stromal Cells Through the Regulation of PI3K/Akt Cascade and Proteasome Activity

AbstractAbstract 3916There is accumulating evidence that green tea extract EGCG [(-)-epigallocatechin-3-gallate] may exert a preventive or a direct anti-tumor effect in several tumor types including chronic lymphocytic leukemia (CLL) and clinical trials with EGCG are already on-going. However, EGCG has a broad spectrum of activities and downstream targets. Therefore, it would be necessary to precisely characterize the key targets of this compound and identify the CLL patients who would most likely profit from EGCG. Therefore, the aim of this study was to evaluate the effect of EGCG on the viability of CLL cells in a well characterized cohort of patients and to get insight into its mechanism of action in CLL.Peripheral blood mononuclear cells (PBMC) of 27 CLL patients were included in this study. Patients were characterized according to the Rai/Binet stage, IgVH mutation status and cytogenetics (13q-del, 11q-del, 17p-del, trisomy-12). The percentage of the leukemic cells (CD19+/CD5+) ranged between 60–98%. CLL cells were exposed to a wide range of concentrations of EGCG (0.1 – 200μM) and cell viability was evaluated by cell titer blue (CTB) assays and FACS analysis after 4 hours, 1, 2 and 3 days. Treatment with EGCG was performed in suspension cultures and under co-culture with primary human bone marrow stromal cells (BMSC).Cell viability assays demonstrated a dose and time dependent decrease in the cell viability after the exposure to EGCG with an IC50 ranging between 50–80μM (25–50μg/ml). A moderate variation in the response to EGCG was observed between patients demonstrating the heterogeneity of the disease. No clear correlation between the in vitro response to EGCG and the clinical background and prognostic markers could be observed in this cohort of patients. Annexin V/propidium iodide (Anx/PI) staining showed that EGCG increased the percentage of early apoptotic (Anx+/PI-) and late apoptotic/necrotic cells (Anx+/PI+). These data suggest that EGCG exerts a pro-apoptotic effect and activates other cell killing mechanisms in CLL cells. The leukemic cells (CD19/CD5) were relatively more sensitive to the compound compared to T cells and monocytes. Co-culture experiments showed that EGCG effectively overcomes the supportive effect of BMSC and induces apoptosis/cell killing in CLL cells. BMSC were less sensitive to the compound and a toxic effect was observed at a concentration of 200 μM or higher. RT-PCR showed a downregulation of the catalytic domain p110a and the regulatory domain p85 of phosphoinositide 3-kinases (PI3K) as well as Bcl-2 and Mcl-1 mRNA expression after exposure to EGCG. Western blotting analysis demonstrated a decrease in the phosphorylation of Akt particularly at pThr308 residue and de-phosphorylation of the tumor suppressor PTEN at pSer380 residue in parallel to the induction of PARP cleavage. In addition, EGCG induced a decrease in the protein expression of the activation marker CD23 and the adhesion molecule CD44. Furthermore, proteasome assays showed that EGCG inhibits the chymotrypsin-like activity within 4 hours of incubation in parallel to induction of early apoptosis. This effect was more remarkable after 24 hours. However, EGCG was less effective in proteasome inhibition compared to Bortezomib.In conclusion, these data demonstrate that EGCG induces cell death in CLL cells through a complex mechanism which may involve the inactivation of PI3K/Akt signaling cascade and inhibition of proteasome activity. The results also point to a potential therapeutic effect of EGCG in CLL which warrants further evaluation. Disclosures:No relevant conflicts of interest to declare.

Molecular Characterization of Chronic Lymphocytic Leukemia Patients with a High Number of Losses in 13q14.

AbstractAbstract 2871Deletion at 13q14 (13q) is the most common genomic aberration in CLL. It is present in more than 50% of cases, and is the sole documented cytogenetic abnormality in 36% of the patients. These latter cases are known to have a more favorable clinical course. However, recent data from our group and others, suggest that patients with CLL and 13q deletion as the only FISH abnormality could have a different outcome depending on the number of cells displaying this aberration. Thus CLL patients with a high number of 13q cells usually had both shorter overall survival and shorter time to first therapy. However, to the best of our knowledge the molecular characteristics of these patients have not been so far analyzed in detail.A total of 102 samples were selected for the study, 32 of which served as a validation cohort. A complete immunophenotypic analysis by flow cytometry and FISH studies were carried out in all cases. The median age was 68 years (range, 35 to 90 years). For the purpose of the study, only samples with one cytogenetic abnormality were included. For the gene expression profile analysis, according to our previous results, two groups of patients with 13q were compared: those in whom 80% or more of cells showed 13q (13qH) and those in whom fewer than 80% of cells showed 13q losses (13qL). The distribution of cases in the study cohort was: 13qH (n=25; 36%), 13qL (n=27; 39%), normal FISH (nCLL, n=8; 11%) and 17p/11q (n= 10; 14%); and in the validation cohort: 13qH (n=7; 22%), 13qL (n=11; 34%) and nCLL (n=9; 28%).Peripheral blood mononuclear cells (PBMCs) were isolated from fresh peripheral blood samples using Ficoll gradient, snapfrozen and stored at 80°C. For the validation cohort, CD19positive B cells were purified by magnetically activated cell sorting (MACS) CD19 MicroBeads resulting in a >98% purity, as analyzed by flow cytometry. CD19positive normal B cells from peripheral blood of five healthy donors served as controls. All samples were hybridized with the Affymetrix Human Exon arrays 1.0 ST.A total of 3 450 genes significantly distinguished 13qH from 13qL patients, defining the 13qH signature. To determine the biological significance of the deregulated genes, a further analysis was carried out, revealing that apoptosis, BCR and NFkB signaling were the most significant affected pathways in 13qH CLL patients. Moreover, 13qH CLL patients were also characterized by a striking overrepresentation of deregulated miRNAs. A total of 15 miRNAs were deregulated in 13qH relative to 13qL patients. HsamiR155 was the most highly upregulated miRNA (Rfold=3.70), while hsamiR223 was the most significantly downregulated (Rfold=0.10). The posttranscriptional regulatory network of miRNA and genes in CLL patients with more than 80% of 13q cells was carried out by analyzing the miRNAmRNA relationships and the pathway analysis demonstrated that B cell receptor signaling, PI3K signaling and NFkB signaling were among the most strongly affected pathways in 13qH patients, highlighting the importance of miRNA regulation in CLL. The influence of other factors with prognostic relevance in CLL, such as IGVH mutational status, was discarded. We also analyzed the gene signature of CLL high risk cytogenetic subgroups in comparison with 13q patients. Surprisingly, our results suggest that some of the biological characteristics of 13qH CLL patients were similar to those of highrisk cytogenetic subgroups, since they share the deregulation of several key signaling pathways. To validate the differences observed between the subgroups of 13q CLL patients and get a visualization of these, we applied the Principal Component Analysis (PCA) in an independent series of patients. The expression pattern of CD19+ cells from CLL patients was notably different from the gene expression profile of CD19+ cells from healthy donors. Thus, CLL patients with a high number of 13q cells can be differentiated based on their expression profile. By contrast, the gene expression of B lymphocytes from 13qL and normal FISH subgroups was similar.Therefore, this study provides new evidences regarding the heterogeneity of 13q deletion in CLL patients. Thus an overexpression of BCR and NFKB patways and as well as a deregulation of the balance between the proliferative and apoptotic signals and miRNA expression are involved in cases with higher percentages of 13q- cells. Disclosures:No relevant conflicts of interest to declare.

Gene expression profiling identifies ARSD as a new marker of disease progression and the sphingolipid metabolism as a potential novel metabolism in chronic lymphocytic leukemia

Several studies demonstrated IGVH mutational status and ZAP70 expression as the most relevant prognostic markers in Chronic Lymphocytic Leukemia (CLL), suggesting the separation of two patient subgroups: with good mutated ZAP70 negative (MTZAP70(-) and poor unmutated ZAP70 positive (UMZAP70(+)) prognosis. We determined the gene expression of B cells in 112 CLL patients divided into three classes: class 1 with MTZAP70(-), class 2 with UMZAP70(+), and class 3 included both UMZAP70(-) and MTZAP70(+). We found LPL, AGPAT2, MBOAT1, CHPT1, AGPAT4, PLD1 genes encoding enzymes involved in lipid metabolism overexpressed in UMZAP70(+). In addition, this study identified ARSD, a gene belonging to the sphingolipid metabolism, as a new gene significantly overexpressed in UMZAP70(+) compared to MTZAP70(-). Western blots confirmed that ARSD protein levels were significantly different between the 3 classes of patients and normal controls. Statistical analysis identified a significant correlation between ARSD and IGVH; however, both ARSD protein level and IGVH were independently associated with the need for therapy of CLL patients. ARSD is a novel prognostic factor as the time to start therapy is shorter in patients with high levels of ARSD protein and sphingolipid metabolism could represent a new biological mechanism in CLL.

P1.32 Prognostic Value of T-Cell Cd38 Expression in B-Chronic Lymphocytic Leukemia

ABSTRACT B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with some patients having an indolent course never needs treatment, while others having rapidly progressive one requires intensive treatment. In recent decades, numerous prognostic markers, such as IgVH mutational status, ZAP-70 and the expression of CD38 on leukemic cells were introduced to screen for patients likely to have progressive course of B-CLL bearing the potential to facilitate risk-adapted treatment strategies. In B-CLL, T cell function is shown to be dysregulated. CD38 has been demonstrated to be an important transmembrane signaling molecule of T cell with a direct effect on its function. The present study was conducted to analyze CD38 expression on T cells by flow cytometry to evaluate its impact on the clinical course of 88 unselected B-CLL patients and correlate it with other risk factors. CD38 expression level on T cells was shown to predict the clinical course of B-CLL in male patients but not in female patients. Male patients showed CD38 expression on T cells in a stage-dependent manner, in contrast to female patients who showed higher expression irrespective to clinical staging. CD38 expression on T cells negatively interacted with treatment-free survival in male patients. Multivariate analysis revealed that CD38 expression level on T cells is an independent prognostic factor in B-CLL male patients. A simultaneous evaluation of CD38 expression on both B-CLL cells and T cells allowed predicting male patient groups with the most favorable prognosis as well as those with the worst.

Cancer testicular antigens as a prognostic markers of chronic lymphocytic leukemia.

6575 Background: CLL is the most common leukemia in adults and has a variable course and prognosis. Clinical staging systems and the use of biomarkers such as cytogenetics and the IGVH mutational status remain the gold standard for stratification. Cancer testis antigens (CTAs) expression analyses have shown prognostic impact in other hematological malignancies such as acute leukemias and multiple myeloma. With the analysis of CTAs we aim to discover additional molecular markers that can help us predict the clinical course and outcome in CLL patients. Methods: We analyzed the expression by RT-PCR of 39 known CTAs including members of the MAGE, GAGE, MAD-CT and SSX CTAs families in a group of 59 CLL patients seen at our institution. The Moffitt Cancer Center Total Cancer Care (TCC) database was used to correlated these findings with relevant clinical and molecular markers such as Rai stage, demographic data, initial WBC count, IGVH mutational status, CD38 expression, cytogenetics by FISH analysis and treatment outcomes. Overall survival (OS) and progression free survival (PFS) were calculated using Kaplan Meier curves with a SPSS statistical software Results: Deletion 13q was the most common gene abnormality in 35/59 (59.3%). 29/59 (49.1%) required at least one line of treatment. MAGE family CTAs were present in 53/59 (83.9%), MAD-CT in 24/59 (40.7%), SSX in 14/59 (23.7%) and GAGE in 12/59(20.3%). 35/59 (59.3%) CLL patients expressed 2 or more MAGE family CTAs. No differences were noted in the Rai staging, initial white blood cell count, B symptoms, extranodal disease, presence of CD38, or unmutated IVGH. The OS was not significantly different among all CTAs families with a tendency towards a better OS those expressing the SSX family (p=0.63). Additionally, CLL patients with single expression of MAD-CT1 (p=0.05) and MAGE-B2 (p=0.038) antigens correlated with improved survival. Conclusions: Patients with SSX family have a trend towards improved survival. When analyzed by individual antigen, MAD-CT1 and MAGE-B2 expression was associated with improved survival. Further studies with a larger number of patients are needed to assess the real value of the expression of CTAs in CLL.

Impact of immunoglobulin heavy chain variable region mutational status on the outcome of patients with chronic lymphocytic leukemia harboring isolated 13q deletion.

6608 Background: Several prognostic factors can predict the course of chronic lymphocytic leukemia (CLL). Among them, the IGVH mutational status and the presence of cytogenetic abnormalities are the strongest predictors of outcome. Mutated IGVH and deletion 13q independently confer a survival advantage. CLL patients with mutated IGVH in combination with deletion 13q have a better prognosis when compared to their unmutated IGVH counterparts. However, there is limited data on the outcome of patients harboring favorable deletion 13q and the unfavorable unmutated IGVH. This study aimed at identifying patients with these two indicators in order to obtain important prognostic information. Methods: We used the Moffitt Cancer Center Total Cancer Care (TCC) database to find patients with a diagnosis of CLL between January 1993 and December 2009. Individual charts were reviewed for demographic data and CLL cytogenetics, including IGVH mutation status and presence of deletion 13q. We analyzed the impact of having deletion 13q in combination with an unmutated IGVH on the overall survival (OS) for this subset of CLL patients using Kaplan Meier curves with SPSS statistical software. Results: 546 patients were identified during the aforementioned time period with a diagnosis of CLL. Median age was 62.5 years. 144 (26.4%) of these patients had IGVH and cytogenetic analysis available. 53 patients had 13q deletion as their sole genetic abnormality. Patients with unmutated IGVH and positive for deletion 13q were 19/53 (35.8%). Patients with mutated IGVH and positive for deletion 13q were 34/53 (64.2%). Patients with mutated IGVH and positive for deletion 13q had an OS of 17 years. While patients with unmutated IGVH and positive deletion 13q had a lower median OS of 12 years (91.2% vs 78.9%, p=0.05). Hazard ratio for patients with IGVH mutated and positive deletion 13q was 0.4, p=0.05. Conclusions: Mutated IGVH appears to be associated with improved OS in patients with isolated 13q deletion when compared to patients with unmutated IGVH and isolated 13q deletion. Further research is needed to assess these mutations in relation to other cytogenetic abnormalities in CLL.

Changes in the apoptotic gene expression profile in CLL patients treated with rituximab combined with cladribine and cyclophosphamide-preliminary results

The study was aimed to investigate modifications of apoptotic gene expression profile by microarray technique in 10 patients with chronic lymphocytic leukemia by treatment with rituximab, cladribine and cyclophosphamide (RCC) according to IGHV mutational status. The TaqMan® Low Density Array for 96 gene transcripts was used. Those modifications followed two distinctive patterns largely overlapping the IGHV mutational status. In the IGHV-mutated group, the expression of many proapoptotic genes increased after treatment as compared to initial value. Our results suggest that RCC drugs may act through influence on the expression of some apoptosis-involved genes dependently on the IGVH mutational status.

Predicting survival in chronic lymphocytic leukemia

There is increasing interest in the use of prognostic markers that may predict survival and guide management in patients diagnosed with the early stages of chronic lymphocytic leukemia (CLL). Currently, the most important traditional prognostic factors include clinical staging, lymphocyte doubling time and β2-microglobulin/thymidine kinase; and the most important novel markers include karyotypic aberrations (typically assessed by FISH probes or CpG oligonucleotide karyotyping) and IgVH mutation status. Although each of these factors have individually shown significant correlations with survival, there is increasing appreciation that the most complete information may be obtained by using a combination of several factors in prognostic normograms or models. In this article, we review the current state-of-the-art with regards to CLL prognostic factors and discuss how they can be applied in the clinic.

High expression of hematopoietic cell specific Lyn substrate-1 (HS1) predicts poor survival of B-cell chronic lymphocytic leukemia patients

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults in western countries. HS1 protein regulates leukemic cell migration and homing, and can indirectly promote disease progression and influence patient survival.The aim of this study was to evaluate HS1 expression in CLL patients in connection with other known prognostic factors and patients’ survival. Methods90 untreated CLL patients were included into the study. The control group consisted of 28 healthy matched people. HS1 detection was performed by western-blotting. Mutational status of IgVH, as well as CD38 and ZAP70 expression was also analyzed. ResultsHS1 expression was significantly higher in CLL patients comparing to controls. Positive correlation was shown between HS1 and: age (p=0.0454), Rai stage (p=0.0412), leukocytosis (p=0.0129) and beta-2-microglobulin (p=0.0342). Patients with lymphocyte doubling time shorter or equal to 6 months had higher expression of HS1. Patients with higher HS1 expression had shorter survival than those with lower HS1 expression (p=0.0329). ConclusionsWe showed, that high HS1 expression predicts poor survival of chronic lymphocytic leukemia patients.

Disruption of TP53 function by Point Mutations and Deletions Is Associated with An Increased Risk of Disease Progression within Previously Treated, Relapsed Chronic Lymphocytic Leukemia Patients

Abstract▪2445▪This icon denotes a clinically relevant abstractChronic lymphocytic leukemia (CLL) patients with a deletion of the TP53 tumor supressor gene located at 17p13 have a poor prognosis in first line chemotherapy regimens. Recent studies indicated somatic TP53 mutations as a prognostic factor in CLL independent of 17p13 deletion status. We aimed to further characterize the prognostic value and the impact of TP53 mutations on progression-free survival (PFS) in the presence and absence of a 17p13 deletion in previously treated and relapsed CLL patients within an international phase III clinical study comparing Fludarabine and Cyclophosphamide with or without Rituximab (FC versus R-FC: REACH trial).We analyzed 457 patients at diagnosis for mutations in the TP53 gene using a combination of a microarray-based resequencing assay (AmpliChip p53 Test, Roche Molecular Systems, USA.) and Sanger sequencing of TP53 exons 2–10. The data were correlated with clinical and biologic markers as well as with interphase fluorescence in situ hybridization (FISH) and with PFS. Association of the clinical data with PFS was assessed by Cox proportional hazard models. To estimate the functional significance of the individual TP53 mutations we used the IARC TP53 database.TP53 mutations (n=60) were detected in 52 of 457 patients (11.4%) and included 42 missense, 4 nonsense, 8 frameshift mutations, 2 in-frame deletions and 4 mutations in splice sites. Among other clinical variables, only 17p13 deletion was associated with TP53 mutations: 27 of 52 TP53 mutated patients had a 17p13 deletion (concordance rate: 52%, Fisher’s test p<0.001). Median PFS for patients with TP53 mutations (n=52, 13 months, HR=1.9 (1.4–2.7), p<0.001) was significantly shorter as compared to patients without TP53 mutations (n=480, 27 months). In a sub-group analysis, chemoimmunotherapy including Rituximab did not significantly improve the PFS of patients with TP53 mutations. Multivariate analysis including treatment arm, Binet stage, age, IGVH mutational status, 17p13 deletion and TP53 mutation status confirmed TP53 mutation status (HR-TP53=1.7 (1.1–2.6), p=0.009) as a prognostic factor for PFS independent of 17p13 deletion status (HR-17p=1.7 (1.1–2.7), p=0.024) and with a similar effect size. The other independent prognostic factors were treatment (HR=0.61 (0.48–0.76), p<0.001), Binet stage (HR=1.64 (1.3–2.1), p<0.001) and IGVH mutational status (HR=2.4 (1.85–3.1), p<0.001). To further dissect the contribution of TP53 mutation and 17p13 deletion on PFS, we considered a multivariate analysis comparing patients with both TP53 mutation and 17p13 deletion (n=28), with only 17p13 deletion (n=9), with a dominant negative TP53 mutation or multiple TP53 mutations (n=8) or with a single TP53 mutation (n=16) against patients without TP53 abnormalities (n=271), adjusted for treatment, Binet stage, age and IGVH mutational status. Patients with a predicted biallelic disruption of TP53 either by a TP53 mutation in combination with a 17p13 deletion (HR: 2.8 (1.8,4.2), p=<0.001) or patients with a dominant negative TP53 mutation as predicted by the IARC TP53 database or multiple TP53 mutations (HR=3.26 (1.5,7.1), p=0.003) had a risk similar in size and which was quite high for disease progression (the reference to calculate the risk, here and in the following, is always the group of patients without TP53 abnormalities). The risk slightly decreased for patients with only a deletion 17p13 (HR=2.2, (1.1–4.3), p=0.021). Very interestingly, single TP53 mutations showed a much lower risk for disease progression (in this case not even significant) (HR=1.61 (0.9–2.8), p=0.084) especially compared to the risk conferred by a biallelic disruption.In this large cohort of previously treated CLL patients, complete disruption of TP53 function (by a combination of a 17p13 deletion and a TP53 mutation, through dominant negative TP53 mutations or through multiple TP53 mutations) was associated with a higher risk for disease progression. Prognosis of patients with a single TP53 mutation was not significantly different from patients without TP53 aberrations. It remains to be shown whether CLL patients with a single TP53 mutation are at a higher risk of acquiring additional mutations of TP53 during disease progression. Prognostic stratification of previously treated CLL patients should include a routine molecular TP53 mutational analysis in addition to deletion analysis of the TP53 locus by FISH. Disclosures:Dufour:Roche: Research Funding. Bohlander:Roche: Research Funding. Spiekermann:Roche: Research Funding. Schneider:Roche: Research Funding. Hiddemann:Roche: Research Funding. Truong:Roche: Employment. Patten:Roche: Employment. Wu:Roche: Employment. Dmoszynska:Mundipharma:; Roche: Honoraria. Robak:Centocor Ortho Biotech Research & Development: Research Funding. Geisler:Roche: Speakers Bureau. Dornan:Genentech: Employment. Lin:Genentech: Employment. Yeh:Genentech: Employment. Weisser:Roche: Employment. Duchateau-Nguyen:Roche: Employment. Palermo:Roche: Employment.