High Cumulative Illness Rating Scale (CIRS) Score in CLL Correlates with Short CLL Telomere Length and Decreased Survival

Abstract The impact of chronic disease on the development and progression of cancer is increasingly recognized. Chronic lymphocytic leukemia (CLL) is a disease of the elderly and many of these patients have multiple comorbidities, which could shorten an individual's life, either directly or by enhancing CLL progression. In normal cells, including buccal cells (BC), it is known that chronic illness and age can shorten telomere length and this is a surrogate marker of overall survival. In the present study, we have examined the relationship between comorbidities and BC telomere length in CLL patients and determined whether these features can predict patient survival and disease aggressiveness. Telomere length in isolated genomic DNA from buccal and CLL cells of 196 CLL patients was measured at the time of diagnosis, using multiplex quantitative real-time PCR. Comorbidities were measured by the Cumulative Illness Rating Scale (CIRS) and CLL aggressiveness by leukemia cell telomere length. The median age of patients at diagnosis was 66 years (range, 39-89). With a median follow-up of 4.86 years (range, 0.05-7.69 years), approximately half the patients have progressed and one quarter have died. The median CIRS score of these patients was 3 (range, 0-12); a score of ≥7 was considered high. In patients with an elevated CIRS score, a direct correlation was found with increasing age (P<0.0001, r=0.42). In addition, independent of the effects of age, an increased CIRS score was found to correlate with poor overall survival (P=0.048, r=0.18). The median BC relative telomere length (T/S) of 2.01 (range, 0.70-5.66) was longer than the median CLL T/S of 0.53 (range, 0.07-2.48). There was no correlation between buccal and CLL telomere lengths (P=0.21). BC telomere lengths shortened with increasing age (P=0.011), but showed no association with markers of CLL disease, survival or high CIRS scores (P=0.08, r=-0.16). Meanwhile, patients with shorter CLL telomeres showed more aggressive disease with unmutated IGHVstatus (P<0.0001), higher Rai stage (P=0.02), shorter lymphocyte doubling time (P=0.004), earlier time to treatment (P<0.0001) and shorter overall survival (P=0.02). More importantly, short CLL telomere lengths occurred independent of increasing age (P=0.47), and significantly correlated with high CIRS scores (P=0.03, r=-0.18). In summary, while BC telomere lengths shorten with age in CLL cases, it is not predictive of survival or comorbidities in CLL. In contrast, independent of age, short CLL telomeres correlate with increasing CIRS scores and both predict poor survival. These results suggest that comorbidities in CLL may affect tumor biology, enhancing disease progression. This finding may partly explain the more aggressive clinical course of CLL in the elderly. Whether altering comorbidities in CLL can influence disease aggressiveness and survival requires further study. Disclosures No relevant conflicts of interest to declare.

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Telomere length predicts progression and overall survival in chronic lymphocytic leukemia: data from the UK LRF CLL4 trial

Leukemia ◽

10.1038/leu.2015.217 ◽

2015 ◽

Vol 29 (12) ◽

pp. 2411-2414 ◽

Cited By ~ 28

Author(s):

J C Strefford ◽

L Kadalayil ◽

J Forster ◽

M J J Rose-Zerilli ◽

A Parker ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Telomere Length ◽

Lymphocytic Leukemia ◽

The Uk ◽

Leukemia Data

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NF-κB in the Vascular Progression of Melanoma

Journal of Clinical Oncology ◽

10.1200/jco.2004.06.047 ◽

2004 ◽

Vol 22 (4) ◽

pp. 617-623 ◽

Cited By ~ 49

Author(s):

Mohammed Kashani-Sabet ◽

Ladan Shaikh ◽

James R. Miller ◽

Mehdi Nosrati ◽

Carlos M.M. Ferreira ◽

...

Keyword(s):

Overall Survival ◽

Cox Regression ◽

Surrogate Marker ◽

Tissue Array ◽

Matched Pair ◽

Vascular Involvement ◽

Tumor Vascularity ◽

Data Set ◽

Cox Regression Analysis ◽

The Impact

Purpose To examine a model of melanoma progression based on vascular factors and the role of NF-κB in the vascular progression of melanoma. Patients and Methods A data set of 526 patients from the University of California San Francisco Melanoma Center with 2 years of follow-up or first relapse was studied. The impact of the presence or absence of various prognostic factors on overall survival of melanoma patients was assessed using Cox regression and Kaplan-Meier analysis. A matched-pair analysis of NF-κB expression was performed in cases with vascular involvement and increased tumor vascularity versus matched controls lacking these factors. Results Cox regression analysis of factors evaluated by the American Joint Committee on Cancer Melanoma Staging Committee reproduced the powerful impact of tumor thickness and ulceration in this data set. With the inclusion of vascular factors such as tumor vascularity and vascular involvement, ulceration was no longer significant in predicting overall survival. By multivariate analysis, vascular involvement and tumor vascularity were the strongest predictors of melanoma outcome. Tumor vascularity seems to be a precursor of both vascular involvement and ulceration. A matched-pair tissue array analysis demonstrated the significant correlation between overexpression of NF-κB–p65 and the development of vascular factors. Conclusion Vascular factors play an important role in the progression of malignant melanoma. Ulceration may be a surrogate marker for the interactions between melanoma and the tumor vasculature. NF-κB seems to play an important role in the development of these factors.

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Comparative Efficacy of First-Line Chemotherapy-Free Combinations in Chronic Lymphocytic Leukemia (CLL): A Network Meta-Analysis

Blood ◽

10.1182/blood-2020-136411 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 25-26

Author(s):

Philip A Haddad ◽

Nowell Ganey ◽

Kevin M. Gallagher

Keyword(s):

Chronic Lymphocytic Leukemia ◽

English Language ◽

Conflicts Of Interest ◽

Residual Disease ◽

Meta Analysis ◽

The Elderly ◽

Lymphocytic Leukemia ◽

First Line ◽

Significant Difference ◽

Anti Cd20

Introduction: Chronic Lymphocytic Leukemia (CLL) is an incurable B-cell malignancy which disproportionately affects the elderly. Although first-line chemoimmunotherapy (CIT) improved CLL clinical outcomes, recent randomized trials revealed superior outcomes with novel chemotherapy-free combinations (CFC) incorporating anti-CD20 monoclonal antibodies and inhibitors of BTK or Bcl-2. So far, these CFC have not been compared head-to-head. We conducted this network meta-analysis to evaluate their relative efficacy to each other. Methods: A review of the medical literature was conducted using online databases. Inclusion criteria consisted of English language; diagnosis of CLL; trials that explored the efficacy of first-line CFC with Obinutuzumab (O), Rituximab (R), Ibrutinib (IB), Acalabrutinib (ACAL), Venetoclax (VEN) compared to standard CIT that included Chlorambucil (CHLOR) with either R or O, Bendamustine+Rituximab, or Fludarabine+ Cyclophosphamide+R; and phase 3 randomized studies reporting responses, progression, death, and adverse (AE) events. A frequentists network meta-analysis was conducted using netmeta package and random-effects model. Results: Five studies comprising a total of 2,272 participants were included. When O-based CFC data was analyzed, only ACAL-O had a significant lower relative risk (RR) of progression and death (P&D). There were no significant differences with respect to overall response rates (ORR), complete remission (CR), minimal residual disease (MRD), or grade >3 adverse events (Grd3+) among O-based CFC. When R-based CFC data was analyzed, IB and IB-R were not different with respect to RR of P&D, ORR, CR, MRD, or Grd3+. When the data was analyzed as CFC versus combined CIT, only ACAL-O was found to be significantly superior to other O- and R-based CFC with respect to RR of P&D. ORR and Grad3+ rates of O- and R-based CFC were not significantly different. While ACAL-O, IB-O, and VEN-O had superior CR and MRD rates compared to other CFC, there were no significant differences among each other. Conclusions: This network meta-analysis is the first to compare and rank first-line CFC therapies in CLL. It indicates that ACAL-O has a superior profile having the lowest RR of P&D without significant difference in Grd3+ among CFC. Disclosures No relevant conflicts of interest to declare.

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Increased Expression of Macrophage Migration Inhibitory Factor (MIF) Receptor CD74 Is Associated with Inferior Outcome in Younger Patients (Pts) with Cytogenetically Normal Acute Myeloid Leukemia (CN-AML): a Cancer and Leukemia Group B (CALGB) Study.

Blood ◽

10.1182/blood.v114.22.1616.1616 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1616-1616 ◽

Cited By ~ 1

Author(s):

Eyal C. Attar ◽

Kati Maharry ◽

Krzysztof Mrózek ◽

Michael D. Radmacher ◽

Susan P. Whitman ◽

...

Keyword(s):

Overall Survival ◽

Cellular Proliferation ◽

Conflicts Of Interest ◽

Advanced Pancreatic Cancer ◽

Disease Free Survival ◽

Prognostic Impact ◽

Free Survival ◽

Expression Levels ◽

The Impact ◽

Disease Free

Abstract Abstract 1616 Poster Board I-642 CD74 is a type II integral membrane protein receptor that binds its ligand MIF to induce phosphorylation of the extracellular signal-regulated kinase-1/2 (ERK-1/2) and drive cellular proliferation via nuclear factor-kappa B (NF-kB) activation. CD74 expression has been identified in human solid tumors, and its expression is associated with adverse prognosis in advanced pancreatic cancer. As CD74 is expressed and NF-kB constitutively activated in myeloblasts, we hypothesized that CD74 expression might also be associated with adverse outcome in AML. To investigate the prognostic impact of CD74 expression in the context of other predictive molecular markers in CN-AML, we assessed CD74 expression levels by Affymetrix HG-U133 Plus 2.0 microarray in 102 younger [<60 years (y)] adults with primary CN-AML, treated on the front-line CALGB 19808 trial with an induction regimen containing daunorubicin, cytarabine, etoposide and, in some cases, the inhibitor of multidrug resistance valspodar, and consolidation with autologous stem cell transplantation. Microarray data were analyzed using the Robust Multichip Average method, making use of a GeneAnnot chip definition file, which resulted in a single probe-set measurement for CD74. At diagnosis, CD74 expression, when assessed as a continuous variable, was significantly associated only with extramedullary disease involvement (P=.006) among clinical features, and with none of the molecular prognostic variables tested, including NPM1, WT1, CEBPA, FLT3 (FLT3-ITD and FLT3-TKD) mutations, MLL partial tandem duplication, or differential BAALC and ERG expression levels. Although CD74 expression levels were not associated with achievement of complete remission (CR; 83% vs 81%), higher levels of CD74 were associated with shorter disease-free survival [DFS; P=.046, hazard ratio (HR) 1.85, 95% confidence interval (CI) 1.12-3.08] and with shorter overall survival (OS; P=.02, HR 1.32, CI 1.04-1.67). In multivariable analyses, higher CD74 expression was independently associated with shorter DFS (P=.045, HR 1.98, CI 1.16-3.40), after adjusting for WT1 mutations (P<.001) and FLT3-TKD (P=.04), and shorter OS (P=.01, HR 1.58, CI 1.11-2.25) after adjusting for FLT3-TKD (P=.02), WT1 mutations (P=.007), BAALC expression levels (P=.02), white blood counts (P=.007), and extramedullary involvement (P=.04). As quartiles 2-4 had similar expression levels distinct from the lowest quartile, to display the impact of CD74 expression levels on clinical outcome only, pts were dichotomized into low (the lowest quartile) and high (the top three quartiles) CD74 expressers. The Kaplan-Meier curves for DFS and OS (Figures 1 and 2) are shown below. In conclusion, our study identifies elevated CD74 expression as associated with adverse prognosis in younger CN-AML pts. Since we previously reported that higher CD74 expression was favorably associated with achievement of CR in AML patients receiving chemotherapy plus bortezomib, an inhibitor of the proteasome and NF-kB (Attar et al., Clin Cancer Res, 2008;14:1446-54), it is possible that in future studies elevated CD74 levels can be used not only for prognostication, but also to stratify CN-AML pts to study of bortezomib-containing chemotherapy regimens. Figure 1 Disease free survival Figure 1. Disease free survival Figure 2 Overall survival Figure 2. Overall survival Disclosures No relevant conflicts of interest to declare.

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TIMP-1 and TIMP-2 Levels Are Directly Correlated with Neoangiogenesis and Are Prognostic Factors in Multiple Myeloma Patients.

Blood ◽

10.1182/blood.v114.22.4866.4866 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4866-4866

Author(s):

Luciana Correa Oliveira de Oliveira ◽

Juliana Alves Uzuelli ◽

Ana Paula Alencar de Lima Lange ◽

Barbara Amelia Aparecida Santana-Lemos ◽

Marcia Sueli Baggio ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Overall Survival ◽

Prognostic Factors ◽

Bone Disease ◽

Cox Regression ◽

Conflicts Of Interest ◽

Newly Diagnosed ◽

Significant Difference ◽

The Impact

Abstract Abstract 4866 Background Multiple myeloma (MM) is an incurable malignant disease, characterized by increased angiogenesis in the bone marrow (BM) microenvironment and aberrant BM metabolism. Matrix metalloproteinases (MMP) are a family of zinc-dependent endopeptidases implicated in tumour progression, invasion, metastasis and angiogenesis, via proteolytic degradation of extracellular matrix. MMPs are inhibited by tissue inhibitors of metalloproteinase (TIMP). Although recent studies have implicated MMP 9 in MM bone disease, little is known about the role of the TIMPs. Objectives a) to compare levels of sRANKL, OPG, MMP-2, MMP-9, TIMP-1, TIMP-2, VEGF, bFGF, microvessel density (MVD) between newly diagnosed MM patients and healthy controls; b) to determine the association of these molecules with disease progression, bone disease and neoangiogenesis and c) to evaluate the impact of these variables on survival. Patients and Methods As of July 2009 38 newly diagnosed and untreated multiple myeloma patients were enrolled in the study. The median age was 61years-old (range 39-91) with 24 (63%) males. Patients were diagnosed and categorized according The International Myeloma Working Group criteria and ISS, respectively. Bone involvement was graded according to standard X-ray: patients with no lesions, or with one/ two bones involved or diffuse osteoporosis were classified as low score, whereas patients with lesions in more than two bones or presence of bone fracture were classified as high score. MMP-2 and MMP-9 were determined by PAGE gelatin zymography from plasma as previously described. MMP-9, TIMP-1 and TIMP-2, OPG and sRANKL concentrations were measured by ELISA. The levels of VEGF, bFGF were obtained using cytometric bead array. Ten healthy volunteers were used as controls. Bone marrow MVD measured in hotspots was evaluated in 26 out of 38 patients at diagnosis and 15 patients with Hodgkin Lymphoma stage IA and IIA (used as controls) by staining immunohistochemically for CD34. Comparisons among groups were analyzed by ANOVA and the correlation by the Spearman's correlation coefficient. Cox regression were performed for overall survival (OS) analysis. Results Patients with MM had elevated TIMP-1, TIMP-2 and OPG values compared with controls. No significant difference was found between plasma sRANKL, pro-MMP2, pro-MMP9 and MMP-9 levels. We found that plasma TIMP-1 levels correlated positively with bFGF, VEGF, MVD, beta-2 microglobulin (B2M) and OPG (r: 0.514, p=0,001, r: 0.350, p=0,031; r: 0.610, p<0.0001; r: 0.760, p<0.0001 and r: 0.701, p<0.0001, respectively) and TIMP-2 levels with bFGF, DMV, B2M and OPG (r: 0.512, p=0.002; r: 0.595, p<0.0001; r: 0.587, p<0.0001 and r: 0.552, p<0.0001, respectively). TIMP-1 and TIMP-2 levels correlated with the ISS stage (p<0.0001, p=0.006, respectively). The only variables that correlated with clinical bone disease staging were hemoglobin, B2M and albumin levels, whereas TIMP-1, TIMP-2, bFGF, VEGF and OPG correlated with DMV. On the univariate analyses, age, gender, proMMP2, TIMP-1, TIMP-2, creatinine, B2M and MVD were significantly associated with overall survival. In Cox regression model, TIMP-1, TIMP-2 and B2M levels remained to be significantly associated with OS. In conclusion, our results suggest that TIMP-1 and TIMP-2 levels are strongly associated with neoangiogenesis and are independent prognostic factors in MM. Disclosures No relevant conflicts of interest to declare.

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High Resolution Array-CGH Provides New Insights Into the Prognosis of Chronic Lymphocytic Leukemia (CLL): Is 8p Loss Worse Than 17p Loss?.

Blood ◽

10.1182/blood.v114.22.2339.2339 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2339-2339

Author(s):

Andrea Rinaldi ◽

Michael Mian ◽

Davide Rossi ◽

Francesco Forconi ◽

Clara Deambrogi ◽

...

Keyword(s):

Cox Regression ◽

Clinical Course ◽

Conflicts Of Interest ◽

Univariate Analysis ◽

Lymphocytic Leukemia ◽

Rank Test ◽

Western World ◽

Clinical Parameters ◽

Log Rank Test ◽

The Impact

Abstract Abstract 2339 Poster Board II-316 BACKGROUND: CLL, the most common adult-onset leukemia in the Western world, has a heterogeneous clinical course. Many advances have led to a better understanding of its pathogenesis and to improvements in treatment strategies, but striking solutions are still missing. We conducted a study to evaluate the impact of genomic aberrations on the clinical course. METHODS: From January 1980 to May 2008, 395 frozen samples of CLL patients, were prospectively collected in four centers. Extracted DNA was analyzed with Affymetrix Human Mapping 6.0 arrays. Normal matched DNA was analyzed for one fourth of the cases. Correlations between minimal common regions (MCR) and clinical parameters were evaluated with the Fisherôs-exact test and their impact on OS with the log-rank test. A p-value after Bonferroni multiple test correction (MTC) (p-adj.) <0.05 was considered as statistically significant. Up to now 266 samples have been analyzed. RESULTS: Analysis of the clinical parameters (CPs) and known risk factors (Rai/Binet, age, doubling time, LDH, beta2, IGVH status, p53 mutations, telomere length, CD38, 11q, 17p) was consistent to previous published series. ZAP70 did not affect the clinical course, likely due inter-laboratories variability. After a median follow up of 53 months, 143/239 (60%) of the patients have started therapy and 63/261 (24%) died. 5-yr OS was 82%. Fisher test between the MCRs and CPs revealed an inverse relation between the presence of trisomy 12 by FISH and del13q14.3, an association between del17p and losses of 8p regions and between CD38 and 12q gain. Before MTC, 46 MCRs had a significant impact on OS and 67. After MTC, 3 regions maintained their role: 8p22 loss (38/248, 15%, p-adj.=0.002, median OS: 26 months vs. 48), 17p13.3-11.2 loss (20/248, 8%, p-adj.=0.001; median OS: 10 months vs. 48). In univariate analysis, the log-rank test among pts with 8p-/17p- (8/248, 3%), 8p- (30/248, 12%), 17p- (12/248, 5%), wild type (198/248, 80%) was statistically significant (p<0.001; see figure). Importantly, none of the analyzed clinical and biological parameters was associated with this aberration. CONCLUSIONS: Loss of 8p22 designated a CLL subgroup with a worse outcome among all patients and in the subset with 17p loss. Our data suggested that this aberration might constitute an independent prognostic factor to be evaluated in independent studies. Results, including a Cox regression model, will be presented on all 395 cases. Disclosures: No relevant conflicts of interest to declare.

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Plasma Circulating Caspase-3 Index as a Biomarker in Patients with Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.4381.4381 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4381-4381

Author(s):

Jean-Marie Bruey ◽

Zeev Estrov ◽

Hagop Kantarjian ◽

Susan O'Brien ◽

Michael Keating ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Normal Control ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Performance Status ◽

Surrogate Marker ◽

Quantitative Measure ◽

Lymphocytic Leukemia ◽

Mutation Status ◽

Control Subjects

Abstract Abstract 4381 Because of the accumulation of lymphocytes and relative paucity of proliferating cells characteristic of B-cell chronic lymphocytic leukemia (CLL), defective apoptosis has been proposed as a key step in the pathogenesis of this disease. Activity levels of the proapoptotic enzyme caspase-3 have been used as a simple, quantitative measure of ongoing apoptosis in various cancers. Here we explored the clinical value of assessing apoptosis levels in patients with CLL, using caspase-3 activity in plasma as a surrogate marker for apoptosis. The study included 194 patients with CLL and 96 normal control subjects. Caspase-3 activity was measured in plasma samples by incubation with the substrate DEVD. Circulating caspase-3 activity was detectable in the plasma of all CLL patients and normal control subjects, but was significantly (P=0.005) lower in CLL patients (median=7.49; range=4.2-19.68 pmol/min/μL) than in controls (median=8.27; range=4.54-34.30 pmol/min/ul. Absolute levels of caspase-3 levels in plasma in CLL did not correlate with any of the laboratory variables examined (WBC, platelets, HGB, B2M), Rai stage, or performance status. To assess the extent of apoptosis in relevance to level of the disease or tumor load, the caspase-3 index was calculated by normalizing plasma caspase-3 activity to the number of circulating lymphocytes in peripheral blood. The circulating caspase-3 index correlated negatively with bone marrow cellularity (P<0.001), spleen size (P= 0.002), and number of sites of enlarged lymph nodes (P<0.001). Interestingly, the circulating caspase-3 index correlated positively with Rai stage (P=0.03, Kruskal-Wallis) but not IgVH mutation status (P=0.74) or performance status (p=0.72). More importantly, higher circulating caspase-3 index values (>8 pmol/min/1000 lymphocytes/ul) were significantly associated with poor overall survival (P=0.005). However, in multivariate analysis incorporating caspase-3 index along with beta-2 microglobulin level and IgVH mutation status showed that caspase-3 was not predictor of survival (p=0.7). In conclusion, apoptosis as determined using plasma caspase-3 activity is low in CLL. However, high circulating caspase-3 activity index values appear to reflect more aggressive disease. Further studies are needed to explore the possibility that this circulating caspase-3 index reflects the proportion of cells that are transformed into larger cells, with consequently higher proliferation and apoptosis rates, in patients with CLL. Disclosures: No relevant conflicts of interest to declare.

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Extensive Intraclonal Diversification in a Subgroup of Chronic Lymphocytic Leukemia Patients with Stereotyped IGHV4-34/IGKV2-30 B cell Receptors: Implications for Ongoing Interactions with Antigen.

Blood ◽

10.1182/blood.v114.22.2337.2337 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2337-2337

Author(s):

Lesley-Ann Sutton ◽

Efterpi Kostareli ◽

Anastasia Hadzidimitriou ◽

Nikos Darzentas ◽

Athanasios Tsaftaris ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Heavy Chain ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

B Cell Receptors ◽

Valuable Insight ◽

Cell Receptors ◽

Mutational Status ◽

The Impact

Abstract Abstract 2337 Poster Board II-314 Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen (Ag) recognition through the clonotypic B cell receptors (BCRs). However, it is still unclear whether Ag involvement is restricted to the malignant transformation phase or whether the putative Ag(s) may continuously trigger the CLL clone. Valuable insight into these issues may be gleaned from the study of intraclonal diversification (ID) within the immunoglobulin (IG) genes through ongoing somatic hypermutation (SHM). Definitive data regarding ID within IG genes in CLL remains limited and conflicting. In the present study we systematically explored the presence of ID within IG genes of CLL, not only at cohort level but also in subgroups defined by BCR stereotypy and IG gene mutational status. We thus conducted a large-scale subcloning study of both IG heavy and light variable genes, in a total of 1496 and 1008 subcloned sequences from 71 and 56 CLL cases, respectively. The analysis was intentionally biased to cases expressing IGHV4-34/IGKV2-30 IGs (subset #4) and IGHV3-21/IGLV3-21 IGs (subset #2) that exhibit distinctive, subset-biased SHM patterns. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and at least 14 colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample (ii) confirmed mutation (CM) - a mutation observed more than once among subcloned sequences from the same sample. Analysis of heavy chain sequences revealed that 40% (28/71) of cases carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, whilst 32% (23/71) of cases carried only UCMs. The remaining 28% (20/71) of cases carried sets of identical IGHV-D-J subcloned sequences. Although most cases showed no or low levels of ID, an intense and, likely, functionally driven ID was evident in selected cases, especially those belonging to subset #4. The distinct ID in subset #4 was statistically significant when compared to all other groups defined by IGHV gene usage and mutation status, BCR stereotypy or heavy chain isotype. Subsequent analysis of the clonotypic light chains revealed that the impact of ID was generally low, with the outstanding exception again relating to subset #4. In fact, of 22 IGKV-J rearrangements exhibiting CMs, 11 (50%) utilized the IGKV2-30 gene and notably 10/11 (91%) of these were expressed by subset #4 cases. In such cases, the expressed IGKV2-30 gene was affected by an active and precisely targeted ID, analogous to their partner IGHV4-34 gene. These findings suggest that the SHM mechanism may continuously operate in certain subsets of CLL patients, particularly those cases expressing stereotyped IGHV4-34/IGKV2-30 BCRs typical of subset #4. In such cases, the observed ID patterns attest to the very precise targeting of the SHM process and may be considered as evidence for a “stereotyped response” to an active, ongoing interaction with Ag(s). Disclosures: No relevant conflicts of interest to declare.

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Aspirin Induces Apoptosis in Human Leukemia Cells Independently of NF-κB and MAPKs through Alteration of the Mcl-1/Noxa Balance.

Blood ◽

10.1182/blood.v114.22.4825.4825 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4825-4825

Author(s):

Ana M Cosialls ◽

Daniel Iglesias-Serret ◽

Maria Piqué ◽

Montserrat Barragán ◽

Antonio F Santidrián ◽

...

Keyword(s):

Conflicts Of Interest ◽

Leukemia Cell ◽

Cell Types ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Human Leukemia ◽

Mrna Levels ◽

Protein Levels ◽

Human Leukemia Cells ◽

Induced Apoptosis

Abstract Abstract 4825 Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in most cell types. We examined the mechanism of aspirin-induced apoptosis in human leukemia cells. Our results show that aspirin induced apoptosis in leukemia Jurkat T cells independently of NF-κB. Although aspirin induced p38 MAPK and c-Jun N-terminal kinase (JNK) activation, selective inhibitors of these kinases did not inhibit aspirin-induced apoptosis. We studied the regulation of Bcl-2 family members in aspirin-induced apoptosis. The mRNA levels of some pro-apoptotic members, such as BIM, NOXA, BMF or PUMA, were induced by aspirin. However, none of these pro-apoptotic proteins increased and the levels of Mcl-1 protein were reduced. Interestingly, in the presence of aspirin the protein levels of Noxa remained high. This alteration of the Mcl-1/Noxa balance was also found in other leukemia cell lines and primary chronic lymphocytic leukemia cells (CLL). Furthermore, in CLL cells aspirin induced an increase in the protein levels of Noxa. Knockdown of Noxa or Puma significantly attenuated aspirin-induced apoptosis. These results indicate that aspirin induces apoptosis through alteration of the Mcl-1/Noxa balance. Disclosures No relevant conflicts of interest to declare.

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Deacetylase Inhibitor Cocktails Provide Striking Synergistic Interactions in Human Leukemia Cells.

Blood ◽

10.1182/blood.v114.22.4404.4404 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4404-4404

Author(s):

Michele Cea ◽

Antonia Cagnetta ◽

Floriana Fruscione ◽

Santina Bruzzone ◽

Gabriele Zoppoli ◽

...

Keyword(s):

Hydroxamic Acid ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Hdac Inhibitors ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Dominant Negative ◽

Myelogenous Leukemia ◽

Leukemia Cells ◽

Human Leukemia

Abstract Abstract 4404 Cancer cells almost invariably exhibit aberrant histone deacetylase (HDAC) activity leading to changes in chromatine structure, altered gene expression, poor differentiation, impaired apoptosis and increased proliferation. Accordingly, virtually all the HDAC inhibitors currently available show some degree of antitumor activity in preclinical cancer models and several of these compounds are currently under investigation or already approved for the treatment of human malignancies. Such is the case of the hydroxamic acid derivative suberoylanilide hydroxamic acid (Vorinostat, Zolinza), approved for the treatment of cutaneous T cell lymphomas. Sirtuins are a large family of deacetylases characterized by a unique, NAD+-dependent enzymatic mechanism. In addition to their established role in metabolism and longevity, recent evidence points to an emerging role for sirtuins in carcinogenesis. In the attempt to identify drug combinations that would increase the activity of traditional HDAC inhibitors we have explored the combination of valproic acid (VA) and butyrate (BU) with the sirtuin inhibitors cambinol and sirtinol in primary B-cell chronic lymphocytic leukemia (B-CLL) cells (n=35), acute myelogenous leukemia (AML) cells (n=10) and leukemia cell lines. Cell viability was assessed by propidium iodide staining and flow cytometry. Combination indices were determined using the median-effect method. In leukemia cells, exposure to sirtuin inhibitors synergistically increased VA and BU mediated cytotoxicity. Conversely, these drugs were poorly active and failed to show any cooperation in healthy cells, including peripheral blood mononuclear cells and fibroblasts, suggesting a cancer-specific mode of action. Similar results were obtained by combining VA or BU with the Nampt inhibitor APO866, which reduces intracellular NAD+ levels and thereby prevents sirtuin activity. Remarkably, SIRT1 and SIRT6 inhibition per se did not seem to account for cell demise upon HDAC inhibition since expression of a dominant negative SIRT1 isoform or RNA interference-mediated SIRT6 silencing failed to increase VA and BU activity. Our data indicate a specific requirement by leukemia cells for sustained sirtuin activity when classical HDACs are inhibited. This feature is suitable to be therapeutically exploited by combining sirtuin inhibitors or APO866 with classical HDAC inhibitors especially for the treatment of hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

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