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1.  Mapping of the IRF8 gene identifies a 3’ UTR variant associated with risk of chronic lymphocytic leukemia but not other common non-Hodgkin lymphoma subtypes 
Background
Our genome-wide association study (GWAS) of chronic lymphocytic leukemia (CLL) identified 4 highly-correlated intronic variants within the IRF8 gene that were associated with CLL. These results were further supported by a recent meta-analysis of our GWAS with two other GWAS of CLL, supporting the IRF8 gene as a strong candidate for CLL risk.
Methods
To refine the genetic association of CLL risk, we performed Sanger sequencing of IRF8 in 94 CLL cases and 96 controls. We then performed fine-mapping by genotyping 39 variants (of which 10 were identified from sequencing) in 745 CLL cases and 1521 controls. We also assessed these associations with risk of other non-Hodgkin lymphoma (NHL) subtypes.
Results
The strongest association with CLL risk was observed with a common SNP located within the 3’ UTR of IRF8 (rs1044873, log additive odds ratio = 0.7, P=1.81×10−6). This SNP was not associated with the other NHL subtypes (all P>0.05).
Conclusions
We provide evidence that rs1044873 in the IRF8 gene accounts for the initial GWAS signal for CLL risk. This association appears to be unique to CLL with little support for association with other common NHL subtypes. Future work is needed to assess functional role of IRF8 in CLL etiology.
Impact
These data provide support that a functional variant within the 3’ UTR of IRF8 may be driving the GWAS signal seen on 16q24.1 for CLL risk.
doi:10.1158/1055-9965.EPI-12-1217
PMCID: PMC3596428  PMID: 23307532
CLL; NHL; SNPs; IRF8; risk locus
2.  Genome-wide Association Study Identifies Multiple Risk Loci for Chronic Lymphocytic Leukemia 
Berndt, Sonja I. | Skibola, Christine F. | Joseph, Vijai | Camp, Nicola J. | Nieters, Alexandra | Wang, Zhaoming | Cozen, Wendy | Monnereau, Alain | Wang, Sophia S. | Kelly, Rachel S. | Lan, Qing | Teras, Lauren R. | Chatterjee, Nilanjan | Chung, Charles C. | Yeager, Meredith | Brooks-Wilson, Angela R. | Hartge, Patricia | Purdue, Mark P. | Birmann, Brenda M. | Armstrong, Bruce K. | Cocco, Pierluigi | Zhang, Yawei | Severi, Gianluca | Zeleniuch-Jacquotte, Anne | Lawrence, Charles | Burdette, Laurie | Yuenger, Jeffrey | Hutchinson, Amy | Jacobs, Kevin B. | Call, Timothy G. | Shanafelt, Tait D. | Novak, Anne J. | Kay, Neil E. | Liebow, Mark | Wang, Alice H. | Smedby, Karin E | Adami, Hans-Olov | Melbye, Mads | Glimelius, Bengt | Chang, Ellen T. | Glenn, Martha | Curtin, Karen | Cannon-Albright, Lisa A. | Jones, Brandt | Diver, W. Ryan | Link, Brian K. | Weiner, George J. | Conde, Lucia | Bracci, Paige M. | Riby, Jacques | Holly, Elizabeth A. | Smith, Martyn T. | Jackson, Rebecca D. | Tinker, Lesley F. | Benavente, Yolanda | Becker, Nikolaus | Boffetta, Paolo | Brennan, Paul | Foretova, Lenka | Maynadie, Marc | McKay, James | Staines, Anthony | Rabe, Kari G. | Achenbach, Sara J. | Vachon, Celine M. | Goldin, Lynn R | Strom, Sara S. | Lanasa, Mark C. | Spector, Logan G. | Leis, Jose F. | Cunningham, Julie M. | Weinberg, J. Brice | Morrison, Vicki A. | Caporaso, Neil E. | Norman, Aaron D. | Linet, Martha S. | De Roos, Anneclaire J. | Morton, Lindsay M. | Severson, Richard K. | Riboli, Elio | Vineis, Paolo | Kaaks, Rudolph | Trichopoulos, Dimitrios | Masala, Giovanna | Weiderpass, Elisabete | Chirlaque, María-Dolores | Vermeulen, Roel C H | Travis, Ruth C. | Giles, Graham G. | Albanes, Demetrius | Virtamo, Jarmo | Weinstein, Stephanie | Clavel, Jacqueline | Zheng, Tongzhang | Holford, Theodore R | Offit, Kenneth | Zelenetz, Andrew | Klein, Robert J. | Spinelli, John J. | Bertrand, Kimberly A. | Laden, Francine | Giovannucci, Edward | Kraft, Peter | Kricker, Anne | Turner, Jenny | Vajdic, Claire M. | Ennas, Maria Grazia | Ferri, Giovanni M. | Miligi, Lucia | Liang, Liming | Sampson, Joshua | Crouch, Simon | Park, Ju-hyun | North, Kari E. | Cox, Angela | Snowden, John A. | Wright, Josh | Carracedo, Angel | Lopez-Otin, Carlos | Bea, Silvia | Salaverria, Itziar | Martin, David | Campo, Elias | Fraumeni, Joseph F. | de Sanjose, Silvia | Hjalgrim, Henrik | Cerhan, James R. | Chanock, Stephen J. | Rothman, Nathaniel | Slager, Susan L.
Nature genetics  2013;45(8):868-876.
doi:10.1038/ng.2652
PMCID: PMC3729927  PMID: 23770605
3.  Immnuophenotypic and Gene Expression Analysis of Monoclonal B Cell Lymphocytosis Shows Biologic Characteristics Associated With Good Prognosis CLL 
Monoclonal B cell lymphocytosis (MBL) is a hematologic condition wherein small B cell clones can be detected in the blood of asymptomatic individuals. Most MBL have an immunophenotype similar to chronic lymphocytic leukemia (CLL), and “CLL-like” MBL is a precursor to CLL. We used flow cytometry to identify MBL from unaffected members of CLL kindreds. We identified 101 MBL cases from 622 study subjects; of these, 82 individuals with MBL were further characterized. Ninety-one unique MBL clones were detected: 73 CLL-like MBL (CD5+CD20dimsIgdim), 11 atypical MBL (CD5+CD20+sIg+), and 7 CD5neg MBL (CD5negCD20+sIgneg). Extended immunophenotypic characterization of these MBL subtypes was performed, and significant differences in cell surface expression of CD23, CD49d, CD79b, and FMC-7 were observed among the groups. Markers of risk in CLL such as CD38, ZAP70, and CD49d were infrequently expressed in CLL-like MBL, but were expressed in the majority of atypical MBL. Interphase cytogenetics was performed in 35 MBL cases, and del 13q14 was most common (22/30 CLL-like MBL cases). Gene expression analysis using oligonucleotide arrays was performed on 7 CLL-like MBL, and showed activation of B cell receptor associated pathways. Our findings underscore the diversity of MBL subtypes and further clarify the relationship between MBL and other lymphoproliferative disorders.
doi:10.1038/leu.2011.117
PMCID: PMC3164475  PMID: 21617698
4.  Common Occurrence of Monoclonal B-cell Lymphocytosis Among Members of High-Risk CLL Families 
British journal of haematology  2010;151(2):152-158.
Summary
Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic haematological condition characterized by low absolute levels of B-cell clones with a surface immunophenotype similar to that of chronic lymphocytic leukaemia (CLL). In the general population, MBL increases with age with a prevalence of 5–9% in individuals over age 60 years. It has been reported to be higher among first-degree relatives from CLL families. We report results of multi-parameter flow cytometry among 505 first-degree relatives with no personal history of lymphoproliferative disease from 140 families having at least two cases of CLL. Seventeen percent of relatives had MBL. Age was the most important determinant where the probability for developing MBL by age 90 years was 61%. MBL clustered in certain families but clustering was independent of the number of known CLL cases in a family. As is the case with CLL, males had a significantly higher risk for MBL than did females (p=0.04). MBL patients had significantly higher mean absolute lymphocyte counts (2.4 × 109/l) and B-cell counts (0.53 × 109/l) than those with a normal B-cell immunophenotype. Our findings show that MBL occurs at a very high rate in high risk CLL families. Both the age and gender distribution of MBL are parallel to CLL, implying a shared inherited risk.
doi:10.1111/j.1365-2141.2010.08339.x
PMCID: PMC2966536  PMID: 20738309
chronic lymphocytic leukaemia; high risk families; monoclonal B-cell lymphocytosis; flow cytometry
5.  Genetic susceptibility variants for chronic lymphocytic leukemia 
Background
There is strong and consistent evidence that a genetic component contributes to the etiology of chronic lymphocytic leukemia (CLL). A recent genome-wide association study (GWAS) of CLL identified 7 genetic variants that increased the risk of CLL within a European population.
Methods
We evaluated the association of these variants, or variants in linkage disequilibrium (LD) with these variants, with CLL risk in an independent sample of 438 CLL cases and 328 controls.
Results
Of these 7 SNPs, 6 had p-trend < 0.05 and had estimated odds ratios (ORs) that were strikingly comparable to those of the previous study. Associations were seen for rs9378805 (OR = 1.47, 95% CI: 1.19, 1.80, p-trend = 0.0003) near IRF4 and rs735665 near GRAMD1B (OR= 1.47; 95% CI: 1.14, 1.89; p-trend = 0.003). However, no associations (P> 0.05) were found for rs11083846, nor were any found for any SNPs in LD with rs11083846.
Conclusions
Our results confirm the previous findings and further support the role of a genetic basis in the etiology of CLL; however, more research is needed to elucidate the causal SNP(s) and the potential manner in which these SNPs or linked SNPs function in CLL pathogenesis.
doi:10.1158/1055-9965.EPI-09-1217
PMCID: PMC2852480  PMID: 20332261
IRF4; CLL; genetic association
6.  Genome-wide association study of follicular lymphoma identifies a risk locus at 6p21.32 
Nature genetics  2010;42(8):661-664.
To identify susceptibility loci for non-Hodgkin lymphoma (NHL) subtypes, we conducted a three-stage genome-wide association study. We identified two variants associated with follicular lymphoma (FL) in 1,465 FL cases/6,958 controls at 6p21.32 (rs10484561, rs7755224, r2=1.0; combined p-values=1.12×10-29, 2.00×10-19), providing further support that MHC genetic variation influences FL susceptibility. Confirmatory evidence of a previously reported association was also found between chronic lymphocytic leukemia/small lymphocytic lymphoma and rs735665 (combined p-value=4.24×10-9).
doi:10.1038/ng.626
PMCID: PMC2913472  PMID: 20639881
7.  N9986: a phase II trial of thalidomide in patients with relapsed chronic lymphocytic leukemia 
Leukemia & lymphoma  2009;50(4):588-592.
We enrolled 28 eligible patients with relapsed chronic lymphocytic leukemia (CLL) to a phase II study of single agent thalidomide (200 mg/d, with dose escalation up to 1000 mg/d over 60 days). The median age was 66 years and 71% were males. Study participants received a median of 2 (range 1–7) prior treatment regimens and 61% had Rai stage 3–4 disease at enrollment. Grade 3 or higher hematologic toxicity was observed in 13 (46%) and 16 (57%) had grade 3 or higher non-hematologic toxicity. Grade 3–4 tumor flare was observed in five (18%) patients. The overall response rate was 11% (3 of 28) with one (4%) complete remission and two (7%) partial remissions. Duration of response for these three responders was 3, 14 and 15 months. Fourteen (50%) patients had stabilisation of disease for a median duration of 8 months (95% CI: 7–16 months). Median time to progression for all 28 patients was 7.3 months. Although thalidomide appears to have modest clinical activity in pretreated/relapsed CLL primarily based on reduction of the absolute lymphocyte count, in our opinion the toxicity profile precludes it from more active investigation in CLL.
doi:10.1080/10428190902777467
PMCID: PMC3928100  PMID: 19373657
CLL; treatment; relapsed; thalidomide; angiogenesis
8.  Phase II study of interleukin-12 for treatment of plateau phase multiple myeloma (E1A96): A trial of the Eastern Cooperative Oncology Group✩ 
Leukemia research  2009;33(11):1485-1489.
The Eastern Cooperative Oncology Group (ECOG) conducted a phase II trial of interleukin-12 (IL-12) for plateau phase multiple myeloma. Patients were initially treated with IL-12 250 ng/kg I.V. daily for 5 days every 3 weeks. The trial was modified due to toxicity after the first 16 patients. IL-12 was given 300 ng/kg subcutaneously twice weekly for 24 weeks. Of 48 eligible patients, there were 4 objective responses (8.3%), all CR. The median survival and progression-free survival were 42.8 and 11.4 months. Unacceptable grade 3 or 4 non-hematologic toxicity (31% with IL-12 subcutaneously and 63% with IL-12 intravenously) was observed.
doi:10.1016/j.leukres.2009.01.020
PMCID: PMC4070590  PMID: 19243818
Multiple myeloma; Interleukin-12; Immunomodulatory therapy
9.  Experimental Designs for Array Comparative Genomic Hybridization (aCGH) Technology 
Cytogenetic and genome research  2013;139(4):250-257.
Array comparative genomic hybridization (aCGH) technology is commonly used to estimate genome-wide copy number variation and to evaluate associations with copy number and disease. Although aCGH technology is well developed and there are numerous algorithms available for estimating copy number, little attention has been paid to the important issue of statistical experimental design. Herein, we review classical statistical experimental designs and discuss their relevance to aCGH technology as well as their importance for down-stream statistical analyses. Furthermore, we provide experimental design guidance for various study objectives.
doi:10.1159/000348815
PMCID: PMC3728659  PMID: 23548696
10.  Quantitative DNA Methylation Analysis Identifies a Single CpG Dinucleotide Important for ZAP-70 Expression and Predictive of Prognosis in Chronic Lymphocytic Leukemia 
Journal of Clinical Oncology  2012;30(20):2483-2491.
Purpose
Increased ZAP-70 expression predicts poor prognosis in chronic lymphocytic leukemia (CLL). Current methods for accurately measuring ZAP-70 expression are problematic, preventing widespread application of these tests in clinical decision making. We therefore used comprehensive DNA methylation profiling of the ZAP-70 regulatory region to identify sites important for transcriptional control.
Patients and Methods
High-resolution quantitative DNA methylation analysis of the entire ZAP-70 gene regulatory regions was conducted on 247 samples from patients with CLL from four independent clinical studies.
Results
Through this comprehensive analysis, we identified a small area in the 5′ regulatory region of ZAP-70 that showed large variability in methylation in CLL samples but was universally methylated in normal B cells. High correlation with mRNA and protein expression, as well as activity in promoter reporter assays, revealed that within this differentially methylated region, a single CpG dinucleotide and neighboring nucleotides are particularly important in ZAP-70 transcriptional regulation. Furthermore, by using clustering approaches, we identified a prognostic role for this site in four independent data sets of patients with CLL using time to treatment, progression-free survival, and overall survival as clinical end points.
Conclusion
Comprehensive quantitative DNA methylation analysis of the ZAP-70 gene in CLL identified important regions responsible for transcriptional regulation. In addition, loss of methylation at a specific single CpG dinucleotide in the ZAP-70 5′ regulatory sequence is a highly predictive and reproducible biomarker of poor prognosis in this disease. This work demonstrates the feasibility of using quantitative specific ZAP-70 methylation analysis as a relevant clinically applicable prognostic test in CLL.
doi:10.1200/JCO.2011.39.3090
PMCID: PMC3397783  PMID: 22564988
11.  Critical Signal Transduction Pathways in CLL 
Receptor tyrosine kinases (RTKs) are cell-surface transmembrane receptors that contain regulated kinase activity within their cytoplasmic domain and play a critical role in signal transduction in both normal and malignant cells. Besides B-cell receptor (BCR) signaling in CLL, multiple RTKs have been reported to be constitutively active in CLL B-cells resulting in enhanced survival and resistance to apoptosis of the leukemic cells induced by chemotherapeutic agents. In addition to increased plasma levels of various types of cytokines/growth factors in CLL, we and others have detected that CLL B-cells spontaneously produce multiple cytokines in vitro which may constitute an autocrine loop of RTK activation on the leukemic B-cells. Moreover, aberrant expression and activation of non-RTKs, for example Src/Syk kinases, induce resistance of the leukemic B-cells to therapy. Based on current available knowledge, we detailed the impact of aberrant activities of various RTKs/non-RTKs on CLL B-cell survival and the potential of using these signaling components as future therapeutic targets in CLL therapy.
doi:10.1007/978-1-4614-8051-8_10
PMCID: PMC3918736  PMID: 24014299
CLL; Signal Transduction; RTK; Non-RTK; Apoptosis; Kinase Inhibitor; Therapy
12.  Autoimmune Cytopenia in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL): Changes in Clinical Presentation and Prognosis 
Leukemia & lymphoma  2009;50(8):1261-1268.
Improved medical care could have altered the clinical presentation and survival of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) complicated by autoimmune cytopenia (AID cytopenia). We reviewed the clinical characteristics, treatment, and outcome of AID cytopenia that was diagnosed in 75 (4.3%) of 1750 CLL patients seen at a single institution over 10 years. Compared to historical reported data, our study shows a lower rate of autoimmune hemolytic anemia (2.3%), and similar rates of immune thrombocytopenia (2.0%) and pure red blood cell aplasia (0.5%). AID cytopenia occurred at all stages of CLL, responded well to treatment, did not alter overall survival, and contributed to death in only 6 (12%) patients. We propose that more sensitive and accurate diagnostic methods for CLL have decreased the perceived prevalence of AID cytopenia and that improvements in management could have increased the survival of these patients.
PMCID: PMC3917557  PMID: 19811329
13.  Statin and Non-steroidal Anti-inflammatory Drug (NSAID) Use In Relation to Clinical Outcome Among Patients with Rai Stage 0 Chronic Lymphocytic Leukemia (CLL) 
Leukemia & lymphoma  2010;51(7):1233-1240.
Statins and nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly prescribed medications. In vitro studies suggest that statins and NSAIDs may have potential as anti-cancer therapies in low grade non-Hodgkin lymphomas including chronic lymphocytic leukemia (CLL) and a recent observational study found statin use was associated with improved event free survival in patients with follicular lymphoma. Other studies have suggested that statins reduce the efficacy of rituximab by inhibiting binding to CD20. We therefore conducted an observational cohort study of 686 patients with newly diagnosed Rai stage 0 CLL to evaluate whether statin or NSAID use was related to their clinical outcome or influenced the efficacy of rituximab therapy. At diagnosis, 136 (20%) patients took statins and 230 (34%) scheduled daily aspirin, ibuprofen, or naproxen. No difference in time to treatment was observed based on statin or NSAID use. Among patients receiving a rituximab containing first-line therapy, no difference in time to salvage treatment was observed based on statin use. Although previous studies suggested statins may improve event free survival among patients with follicular lymphoma, we find no impact of statins on time to initial therapy in this large study of patients with Rai stage 0 CLL. The in vitro observation that statins reduce rituximab efficacy does not appear to have clinical significance in CLL care.
doi:10.3109/10428194.2010.486877
PMCID: PMC3913168  PMID: 20496995
15.  Phase 2 Trial of Daily, Oral Polyphenon E in Patients with Asymptomatic, Rai Stage 0-II Chronic Lymphocytic Leukemia(CLL) 
Cancer  2012;119(2):363-370.
OBJECTIVES
To follow-up the results of phase I testing by evaluating the clinical efficacy of the green tea extract Polyphenon E for patients with early stage chronic lymphocytic leukemia(CLL).
METHODS
Previously untreated patients with asymptomatic, Rai stage 0-II CLL and an absolute lymphocyte count(ALC) ≥10 ×109/L were eligible for this phase II trial. Polyphenon E with a standardized dose of epigallocatechin-3-gallate(2000 mg per dose) was administered twice daily.
RESULTS
Forty-two patients received Polyphenon E 2000 mg twice daily for up to 6 months. Among these, 29 (69%) had Rai stage I-II disease. Patients received a median of 6 cycles of treatment(range: 1-6). The most common grade 3 side effects were transaminitis (n=1), abdominal pain(n=1) and fatigue(n=1). Clinical activity was observed with 13(31%) patients experiencing a sustained ≥20% reduction in ALC and 20 of 29(69%) patients with palpable adenopathy experiencing at least a 50% reduction in the sum of the products of all nodal areas. EGCG plasma levels after 1 month of therapy correlated with reductions in lymphadenopathy (correlation 0.44; p=0.02). Overall, 29(69%) patients fulfilled the criteria for a biologic response with either a sustained ≥20% decline in ALC and/or a ≥30% reduction in the sum of the products of all nodal areas at some point during the 6 months of active treatment.
CONCLUSION
Daily oral EGCG in the Polyphenon E preparation was well tolerated by CLL patients in this phase II trial. Durable declines in ALC and/or lymphadenopathy were observed in the majority of patients.
doi:10.1002/cncr.27719
PMCID: PMC3902473  PMID: 22760587
16.  Age at Diagnosis and the Utility of Prognostic Testing in Patients with Chronic Lymphocytic Leukemia (CLL) 
Cancer  2010;116(20):4777-4787.
PURPOSE
To analyze the survival of CLL patients relative to age-matched individuals in the general population and determined the age-stratified utility of prognostic testing.
METHODS
All 2487 patients diagnosed with CLL between January 1995 and June 2008 and cared for in the Mayo Division of Hematology were categorized by age at diagnosis and evaluated for differences in clinical characteristics, time to first treatment(TFT), and overall survival(OS).
RESULTS
Among Rai stage 0 patients, survival was shorter than the age-matched general population for patients age<55 years(p<0.001), 55-64 years(p<0.001), and 65-74 years(p<0.001) but not those age≥75 at diagnosis(p=NS). CD38, IGHV mutation, and ZAP-70 each predicted TFT independent of stage for all age groups(all p <0.04) but had less value for predicting OS, particularly as age increased. IGHV and FISH predicted OS independent of stage for patients
CONCLUSIONS
Survival of CLL patients age<75 is shorter than the age-matched general population regardless of disease stage. Among patients age<75, the simple combinations of stage and IGHV or stage and FISH identifies those with excess risk of death relative to the age-matched population. Although useful for predicting TFT independent of stage for patients of all ages, prognostic testing had little utility for predicting OS independent of stage among patients age≥75.
doi:10.1002/cncr.25292
PMCID: PMC3902481  PMID: 20578179
Cancer  2011;118(7):1827-1837.
Background
The impact of physicians’ disease-specific expertise on patient outcome is unknown. While previous studies suggest a survival advantage for cancer patients cared for at high volume centers, these observations may simply reflect referral bias or better access to advanced technologies, clinical trials, and multidisciplinary support at large centers.
Methods
We evaluated time to first treatment(TTFT) and overall survival(OS) of patients with newly diagnosed chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL) at a single academic center based on whether they were cared for by a hematologist/oncologist who sub-specializes in CLL(CLL hematologist) or a hematologist/oncologist with expertise in other areas(non-CLL hematologist).
Results
Among 1309 newly diagnosed patients with CLL cared for between 1999–2009, 773(59%) were cared for by CLL hematologists and 536 were cared for by non-CLL hematologists. Among early stage patients(Rai 0-I), median TTFT(9.2 vs. 6.1 years; p<0.001) and OS(10.5 years vs. 8.8 years; p<0.001) were superior for patients cared for by CLL hematologists. For all patients, OS was superior for patients cared for by CLL hematologists(10.5 years vs. 8.4 years; p=0.001). Physician’s disease-specific expertise remained an independent predictor of OS after adjusting for age, stage, sex, and lymphocyte count. Patients seen by a CLL hematologist were also more likely participate in clinical trials(48% vs. 16%; p<0.001).
Conclusion
Physician disease-specific expertise appears to influence outcome in patients with CLL. To the greatest extent possible, patients should be cared for by a hematologist/oncologist expert in the care of their specific malignancy. When not possible, practice guidelines developed by disease-specific experts should be followed.
doi:10.1002/cncr.26474
PMCID: PMC3893049  PMID: 22009554
chronic lymphocytic lymphoma(CLL); small lymphocytic lymphoma (SLL); prognosis; physician expertise
Purpose
Chronic Lymphocytic Leukemia (CLL) is incurable with current chemotherapy treatments. Curcumin (diferuloylmethane), an active ingredient in the spice turmeric, inhibits tumor metastasis, invasion, and angiogenesis in tumor cell lines. We evaluated the effects of curcumin on the viability of primary CLL B-cells and its ability to overcome stromal mediated protection.
Experimental Design
The in vitro effect of curcumin on primary CLL B-cells was evaluated using FACS analysis and Western blotting. For some experiments, CLL B-cells were co-cultured with human stromal cells to evaluate the effects of curcumin on leukemia cells cultured in their micro-environment. Finally, the effect of curcumin in combination with the green tea extract EGCG was evaluated.
Results
Curcumin induced apoptosis in CLL B-cells in a dose-dependent (5–20 μM) manner and inhibited constitutively active pro-survival pathways including STAT3, AKT and NF-κB. Moreover, curcumin suppressed expression of the anti-apoptotic proteins Mcl-1 and XIAP, and up-regulated the pro-apoptotic protein BIM. Co-culture of CLL B-cells with stromal cells resulted in elevated levels of STAT3, increased expression of Mcl-1, XIAP and decreased sensitivity to curcumin. When curcumin was administered simultaneously with EGCG, antagonism was observed for most patient samples. In contrast, sequential administration of these agents led to substantial increases in CLL B-cell death and could overcome stromal protection.
Conclusions
Curcumin treatment was able to overcome stromal protection of CLL B-cells on in vitro testing and to synergize with EGCG when administered in a sequential fashion. Additional evaluation of curcumin as a potential therapeutic agent for treatment of CLL appears warranted.
doi:10.1158/1078-0432.CCR-08-1511
PMCID: PMC3893060  PMID: 19228728
PLoS ONE  2013;8(12):e83830.
CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.
doi:10.1371/journal.pone.0083830
PMCID: PMC3871531  PMID: 24376763
British journal of haematology  2012;159(5):572-576.
Summary
A recent meta-analysis of three genome-wide association studies of chronic lymphocytic leukaemia (CLL) identified two common variants at the 6p21.31 locus that are associated with CLL risk. To verify and further explore the association of these variants with other non-Hodgkin lymphoma (NHL) subtypes, we genotyped 1196 CLL cases, 1699 NHL cases, and 2410 controls. We found significant associations between the 6p21.31 variants and CLL risk (rs210134: P=0.01; rs210142: P=6.8×10−3). These variants also showed a trend towards association with some of the other NHL subtypes. Our results validate the prior work and support specific genetic pathways for risk among NHL subtypes.
doi:10.1111/bjh.12070
PMCID: PMC3614403  PMID: 23025533
CLL; NHL; SNPs; BAK1; risk locus
British journal of haematology  2008;141(5):10.1111/j.1365-2141.2008.07070.x.
Patients with CLL have a variable clinical course. Identification of modifiable characteristics related to CLL-specific survival may provide opportunities for therapeutic intervention. The absolute number of T-cell and natural kill(NK)-cells was calculated for 166 consecutive patients with CLL evaluated by flow cytometry at Mayo Clinic <2 months of diagnosis. The size of the T-cell/NK-cell compartment relative to the size of the malignant monoclonal B-cell(MBC) compartment was evaluated by calculating NK:MBC and T:MBC ratios. Patients exhibited substantial variation in the absolute number of T and NK-cells as well as T:MBC and NK:MBC ratios at diagnosis. Higher T:MBC and NK:MBC ratios were observed among patients with early stage and mutated IgVH genes(all P<0.0003). As continuous variables, both T:MBC ratio(p value=0.03) and NK:MBC ratio(p value=0.02) were associated with time to treatment(TTT). On multivariate Cox modeling including stage, CD38, absolute MBC count, NK:MBC ratio, and T:MBC ratio, the independent predictors of TTT were stage, T:MBC ratio, and NK:MBC ratio. These findings suggest measurable characteristics of the host immune system relate to the rate of disease progression in patients with newly diagnosed CLL. These characteristics can be modified and continued evaluation of immunomodulatory drugs, vaccination strategies, and cellular therapies to delay/prevent disease progression are warranted.
doi:10.1111/j.1365-2141.2008.07070.x
PMCID: PMC3840945  PMID: 18384436
CLL; prognosis; immune system; T-cells; natural killer cells
Background
Non-Hodgkin lymphoma (NHL) is a malignancy of lymphocytes, and there is growing evidence for a role of germline genetic variation in immune genes in NHL etiology.
Methods
To identify susceptibility immune genes, we conducted a 2-stage analysis of single nucleotide polymorphisms (SNPs) from 1,253 genes using the Immune and Inflammation Panel. In Stage 1, we genotyped 7,670 SNPs in 425 NHL cases and 465 controls, and in Stage 2 we genotyped the top 768 SNPs on an additional 584 cases and 768 controls. The association of individual SNPs with NHL risk from a log-additive model was assessed using the Odds Ratios (ORs) and 95% confidence intervals (CI).
Results
In the pooled analysis, only the TAP2 coding SNP rs241447 (MAF=0.26; Thr655Ala) at 6p21.3 (OR=1.34, 95%CI 1.17-1.53) achieved statistical significance after accounting for multiple testing (p=3.1 × 10−5). The TAP2 SNP was strongly associated with follicular lymphoma (FL, OR=1.82, 95%CI 1.46-2.26; p=6.9 × 10−8), and was independent of other known loci (rs10484561 and rs2647012) from this region. The TAP2 SNP was also associated with diffuse large B-cell lymphoma (DLBCL, OR=1.38, 95% CI 1.08-1.77; p=0.011), but not chronic lymphocytic leukemia (OR=1.08; 95% CI 0.88-1.32). Higher TAP2 expression was associated with the risk allele in both FL and DLBCL tumors.
Conclusion
Genetic variation in TAP2 was associated with NHL risk overall, and FL risk in particular, and this was independent of other established loci from 6p21.3.
Impact
Genetic variation in antigen presentation of HLA class I molecules may play a role in lymphomagenesis.
doi:10.1158/1055-9965.EPI-12-0696
PMCID: PMC3467356  PMID: 22911334
genetics; non-Hodgkin lymphoma; immune function; single nucleotide polymorphisms
Cancer genetics and cytogenetics  2010;203(2):141-148.
Five laboratories in the Chronic Lymphocytic Leukemia (CLL) Research Consortium (CRC) investigated standardizing and pooling of fluorescence in situ hybridization (FISH) results as a collaborative research project. This investigation used fixed bone marrow and blood cells available from previous conventional cytogenetic or FISH studies in two pilot studies, a one-day workshop, and proficiency test. Multiple FISH probe strategies were used to detect 6q-, 11q-, +12, 13q-, 17p-, and IGH rearrangements. Ten specimens were studied by participants who used their own probes (pilot study 1). Of 312 FISH interpretations, 224 (72%) were true-negative, 74 (24%) true-positive, 6 (2%) false-negative, and 8 (3%) false-positive. In pilot study no. 2, each participant studied two specimens using identical FISH probe sets to control for variation due to probe sets and probe strategies. Of 80 FISH interpretations, no false interpretations were identified. At a subsequent workshop, discussions produced agreement on scoring criteria. The proficiency test that followed produced no false-negative results and 4% (3/68) false-positive interpretations. Interpretation disagreements among laboratories were primarily attributable to inadequate normal cutoffs, inconsistent scoring criteria, and the use of different FISH probe strategies. Collaborative organizations that use pooled FISH results may wish to impose more conservative empiric normal cutoff values or use an equivocal range between the normal cutoff and the abnormal reference range to eliminate false-positive interpretations. False-negative results will still occur, and would be expected in low-percentage positive cases; these would likely have less clinical significance than false positive results. Individual laboratories can help by closely following rigorous quality assurance guidelines to ensure accurate and consistent FISH studies in their clinical practice and research.
doi:10.1016/j.cancergencyto.2010.08.009
PMCID: PMC3763815  PMID: 21156226
Cancer genetics and cytogenetics  2010;203(2):161-168.
To better understand the implications of genomic instability and outcome in B-cell CLL, we sought to address genomic complexity as a predictor of chemosensitivity and ultimately clinical outcome in this disease. We employed array-based comparative genomic hybridization (aCGH), using a one-million probe array and identified gains and losses of genetic material in 48 patients treated on a chemoimmunotherapy (CIT) clinical trial. We identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14 and 17. Higher genomic complexity, as a mechanism favoring clonal selection, was associated with shorter progression-free survival and predicted a poor response to treatment. Of interest, CLL cases with loss of p53 surveillance showed more complex genomic features and were found both in patients with a 17p13.1 deletion and in the more favorable genetic subtype characterized by the presence of 13q14.1 deletion. This aCGH study adds information on the association between inferior trial response and increasing genetic complexity as CLL progresses.
doi:10.1016/j.cancergencyto.2010.09.003
PMCID: PMC3026606  PMID: 21156228
CLL; aCGH; genotype; prognosis

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