Survival of chronic lymphocytic leukemia (CLL) cells requires sustained activation of the antiapoptotic PI-3-K/Akt pathway, and many therapies for CLL cause leukemia cell death by triggering apoptosis. Blood lipoprotein particles are either pro- or antiapoptotic. High-density lipoprotein particles are antiapoptotic through sphingosine-1-phosphate receptor 3-mediated activation of the PI-3-K/Akt pathway. Apolipoprotein E4 (apoE4)–very low density lipoproteins (VLDL) increase apoptosis, but the apoE2-VLDL and apoE3-VLDL isoforms do not. As increased B-cell apoptosis favors longer survival of CLL patients, we hypothesized that APOE4 genotype would beneficially influence the clinical course of CLL. We report here that women (but not men) with an APOE4 genotype had markedly longer survival than non-APOE4 patients. VLDL is metabolized to low-density lipoprotein through lipoprotein lipase. Higher levels of lipoprotein lipase mRNA in these CLL patients correlated with shorter survival. The beneficial effect of APOE4 in CLL survival is likely mediated through APOE4 allele-specific regulation of leukemia cell apoptosis. The APOE allele and genotype distribution in these CLL patients is the same as in unaffected control populations, suggesting that although APOE genotype influences CLL outcome and response to therapy, it does not alter susceptibility to developing this disease.
Decreased apoptotic death of CD5+ B lymphocytes contributes to their increased abundance in blood, producing the clinical entity chronic lymphocytic leukemia (CLL). The clinical progression of this disorder is often indolent, with a median survival over 20 years from the time of diagnosis to death.1, 2 CLL is characterized by the overexpression of antiapototic proteins such as bcl-2, and commonly employed therapies in CLL increase apoptotic cell death.1, 2 Activation of the phosphatidylinositol-3-kinase/Akt (PI-3-K/Akt) pathway in CLL cells inhibits apoptotic death.3 Lipoprotein particles in sera also modulate apoptotic cell death.4, 5 High-density lipoprotein particles, interacting through a sphingosine-1-phosphate receptor 3 (S1P3), inhibit apoptosis by activating the PI-3-K/Akt pathway.4 Very low density lipoprotein (VLDL) particles containing apolipoprotein E4 (apoE4) (but not the apoE3 or apoE2 isoforms) inhibit this pathway. ApoE4 VLDL cell surface-binding recruits the phosphoinositol phosphatases sh3-containing inositol polyphosphate-5-phosphatase-2 (SHIP-2) to the plasma membrane causing a decrease in phosphatidylinositol-3,4,5-trisphosphate (PIP3) and subsequent reduction in phosphorylation of Akt. These events result in increased apoptosis.4 As APOE4-VLDL increases apoptosis, and as increased B-cell apoptosis would likely favor longer survival in CLL, we hypothesized that an APOE4 genotype would beneficially influence the clinical course of CLL.
Materials and methods
Patients with CLL were recruited from the Duke University and VA Medical Centers from July 1999 through August 2006 as noted before.6 The 183 patients studied here (from the cohort of 190 patients we reported earlier6) were those on whom we had sufficient DNA to genotype for APOE. Fifty-eight patients (32%) were from the Durham VA Medical Center, and 125 patients (68%) from Duke University Medical Center. One hundred and thirty-three patients (73%) were male. Diagnosis and staging of CLL, and decisions regarding initiation of treatment were determined according to NCI Working Group criteria.6, 7 The length of time from diagnosis to death from any cause was defined as overall survival, and the length of time to initiation of treatment from the date of diagnosis was defined as the time to treatment (TTT). All subjects had not received CLL therapy for at least 4 weeks before blood was sampled, and all patients gave informed consent according to protocols approved by the VA and Duke University Institutional Review Boards.
Blood anticoagulated with sodium heparin was processed to enrich the CLL cells by negative selection using monoclonal antibodies as we did before.6, 8 The enriched CLL cells contained 0.9±0.1% (mean±s.e.m.) T cells and 3.4±0.6% CD19+/CD5− cells (‘normal’ B cells). Purified cells were immunophenotyped on the day of isolation, and some were frozen for later analyses. Doubling times, CLL cell phenotypes including CD38 and Zap-70 assessment, IgVH mutation status and FISH analyses were performed as we have done before.6, 8
APOE genotyping was performed as previously described.9 We assayed lipoprotein lipase (LPL) mRNA by quantitative reverse transcriptase-PCR using TaqMan premade primers (Applied Biosystems, Foster City, CA, USA) for LPL and β-actin (ACAB) genes. RNA was isolated as before,10 and cDNA was synthesized with the high-capacity archive kit (Applied Biosystems) using a minimum of 50 ng RNA. cDNA was amplified using the TaqMan Universal 2X PCR mix (Applied Biosystems). Standard semilogarithmic curves for each LPL and ACAB determination, correlating RNA concentration and Ct values, were constructed for each experiment using a standard preparation of highly purified RNA obtained from pheresis-isolated lymphocytes.10 LPL values were normalized using its corresponding ACAB value. All samples were determined in duplicate. We dichotomized LPL values with receiver-operator-characteristics (ROC) curve analysis using ‘good’ and ‘poor’ prognosis groups. ‘Good’ prognosis was defined as not requiring treatment for >5.5 years from diagnosis (the 75th percentile for TTT), and ‘bad’ prognosis was defined as requiring treatment within <2.6 years from diagnosis (the 50th percentile for TTT). The ROC-determined LPL cutoff value was 6.2 units, with an area under the curve of 0.73. Comparisons of clinical and laboratory parameters between groups were done using the Wilcoxon's test and the χ2 test as appropriate. Survival and time-to-treatment Kaplan–Meier data were analyzed using the log rank test. A two-sided alpha of 0.05 was used for all tests.
We analyzed a cohort of 183 CLL patients (50 females and 133 males) followed in the Durham VA and Duke University Medical Centers (Table 1). Analysis of this cohort by gender revealed a profound survival benefit for female patients with an APOE4 genotype (that is, possessing either one or two APOE4 alleles) (P=0.02) (Figure 1a), but there was no significant benefit for males (Figure 1b). For the overall cohort (females and males together), APOE4 patients had longer survival than those with no APOE4 alleles (that is, a non-APOE4 genotype) (Figure 2a), but this difference for the population was not statistically significant (P=0.09). No statistically significant differences in survival were observed in any of these populations between APOE2 genotype and non-APOE2 genotype patients, or between APOE3 genotype and non-APOE3 genotype patients (data not shown). The long survival in APOE4 CLL patients is contrary to that of the general population in which APOE4 genotype increases the risk for atherosclerosis and death from vascular disease.11
Chronic lymphocytic leukemia is an incurable disease, and the decision to treat this leukemia is based on a variety of factors including decline in other blood elements (development of anemia or thrombocytopenia), increasing lymphocyte count rates (for example, a lymphocyte doubling time of less than 1 year) and systemic side effects such as fever and weight loss.7 TTT from diagnosis therefore reflects the rate of disease progression. The presence or absence of an APOE4 allele did not alter TTT in females (P=0.29) (Figure 1c) or in males (P=0.42) (Figure 1d) separately or in the combined cohort of males and females (P=0.98) (Figure 2b). Of the APOE4 women, 7 of 12 received chemotherapy and none of 12 died. Of the non-APOE4 women, 22 of 38 received chemotherapy and 9 of 38 died. The survival and TTT for all men and women in the entire cohort was equivalent (Figure 3).
The APOE genotype distribution of the CLL patient population did not differ from that of the general population12 (Figure 4). This was true when analyzed for the overall population and for women or men. For example, 17.6% of the women in the general population reported previously12 were of APOE4 genotype, and 17.1% of the women in our CLL patients were of APOE4 genotype. These data suggest that although APOE genotype influences survival with CLL, it does not influence the risk of developing this disease.
Lipoprotein lipase is a secreted enzyme that hydrolyzes phospholipids in VLDL particles, thereby increasing serum triglycerides and converting VLDL particles to low-dendity lipoprotein (LDL) particles. In agreement with earlier publications,13, 14, 15 higher levels of leukemia cell LPL mRNA were significantly associated with a shorter survival and shorter TTT in the analysis of males and females in our CLL cohort independent of APOE status (Figures 5a–d), but this was statistically significant only in males (P=0.003 for survival and 0.005 for TTT). Likewise, in analyzing the entire cohort of males and females together, those with high LPL mRNA levels had a significantly worse survival (P=0.002) and TTT (P=0.002) (Figures 2c and d).
We next analyzed survival of patients with CLL according to both APOE genotype and CLL cell LPL mRNA level (Figures 6a–c). This co-analysis was possible in 110 of the 183 patients for whom enough mRNA was available for LPL analysis. APOE4 genotype patients with either low LPL or high LPL had much better survival (100%) than the non-APOE4 patients with either low LPL or high LPL. This was apparent when the population was considered as a whole (Figure 6a), for females alone (Figure 6b) or for males alone (Figure 6c). These differences were statistically significant for the group as a whole (P=0.007) and for men alone (P=0.003). These results suggest that APOE4 genotype has a stronger effect on survival than does LPL level.
Clinical stage, lymphocyte doubling time, immunoglobulin IgVH mutation status, cytogenetic abnormalities and leukemia cell CD38 and Zap-70 expression are significantly associated with survival in CLL.1, 2, 6, 16, 17, 18 Overall, women were of lower stage than men (P=0.02). Women with CLL and an APOE4 genotype compared with the non-APOE4 women patients were less likely to have Zap-70-positive cells (P=0.03), had a higher hematocrit at the time of diagnosis (P=0.009) and had fewer deaths (P=0.02) (Table 1). There were no differences in CD38 expression, IgVH mutational status and cytogenetic abnormalities between women with and without the APOE4 genotype. High levels of LPL mRNA were significantly associated with unmutated IgVH status (P<0.0001), CD38 positivity (P=0.002), Zap-70 positivity (P=0.0006), high serum lactate dehydrogenase (LDH) (P=0.009) and higher (worse) scores on a CLL prognostic index scale6 (P=0.003) (Table 2). High LPL mRNA levels were also significantly associated with the cytogenetic abnormalities 17p13 del (P=0.02) and trisomy 12 (P=0.02), whereas low levels of LPL mRNA were significantly associated with the 13q14 del abnormality (P=0.04). APOE4 individuals tended to have lower LPL levels than non-APOE4 individuals, but the differences were not statistically significant (Table 1).
We observe here that the APOE4 genotype is associated with markedly increased survival in female CLL patients, but not with the length of time before treatment is required (TTT). These observations suggest that the APOE4 genotype may enhance responses to therapy. Therapies commonly employed in CLL trigger apoptosis of these cells.2, 19 As apoE4-VLDL increases the apoptosis of endothelial cells initiated by withdrawing growth factors,4 it may similarly increase apoptosis of CLL cells exposed to therapeutic drugs. Although our observations are striking and highly significant in this cohort of patients, they need confirmation in an independent cohort of patients.
The APOE4 allele specificity reported here could result from apoE isoform-selective binding of VLDL to cell surface receptors. In endothelial cells, apoE4-VLDL is proapoptotic through its interaction with an as yet unidentified cell surface receptor that is inhibited by the receptor-associated protein. Receptor-associated protein inhibits binding of apoE both to LDL-family receptor members and to heparan sulfate proteoglycans on the cell surface.5, 20 ApoE4-VLDL binding to CLL cells may enhance leukemia cell apoptosis initiated by various means (for example, chemotherapy) and improve overall survival.
Elevated LPL mRNA or LPL protein levels are associated with poorer prognosis in CLL.13, 14, 15 LPL is a secreted lipase that binds heparan sulfate proteoglycans on the cell surface. Furthermore, LPL binds to VLDL particles (that also bind cell surface heparan sulfate proteoglycans) and hydrolyzes VLDL triglycerides (Figure 7).21 LPL thereby converts VLDL particles to LDL particles and increases serum fatty acids. Elevated LPL enzymatic activity would therefore increase the rate of conversion of VLDL to LDL. The deleterious effects of elevated LPL activity on survival in CLL may be due to the reduced abundance of apoE4-VLDL particles, a condition that would lead to decreased apoptosis.
Several groups have reported that women have a slightly longer survival than men in CLL (see Molica22 for review), but this was not the case in our cohort. We did note, however, that the effect of the APOE4 allele on survival is much more pronounced in women, whereas the effect of LPL levels on survival is more pronounced in men. Women with CLL generally present with fewer unfavorable clinical features and respond better to treatment.22 The reasons for these gender differences are not known. In our cohort, 92% of the women with CLL and an APOE4 genotype were postmenopausal at the time of diagnosis, but most were taking hormonal replacement therapy. Of the 50 women (range of age at diagnosis 42–85 years), 45 were postmenopausal, and 23 of 25 (on whom information was available) were receiving hormonal replacement therapy. Estrogens can be cytotoxic for CLL cells in vitro,23 and diethylstilbestrol treatment of prostate cancer in patients with CLL has been reported to reduce blood CLL cell counts.24 Several physiological metabolites of endogenous estrogen (including 2-methoxyestradiol) induce apoptosis of leukemia cells by inactivating Akt.25 Estrogen receptor agonists stimulate PIP3 synthesis and enhances Akt phosphorylation in endothelial cells.26 We have shown that Akt phosphorylation is inhibited by APOE4-VLDL.4 Estrogen regulates APOE expression.27 Androgen interacts with apolipoprotein genotype, protecting against APOE4-induced cognitive deficits.28 APOE genotype alters lipoprotein particle distribution and number and triglyceride metabolism, and gender differences in these effects have been reported.29, 30 These observations suggest that the female-specific protective effect of the APOE4 genotype on survival in CLL could relate to several mechanisms including modulation of CLL cell apoptosis by estrogen through the apoE/Akt pathway.
APOE genotyping of patients with CLL may provide important clinical prognostic information, particularly in women. Most importantly, the allele-specific influence of APOE on disease progression may provide important new insights into the mechanisms of disease and response to therapy. Although the APOE4 allele is associated with improved survival of women with CLL, the mechanism of this effect is not known. The apoE4 isoform itself, through isoform-specific interactions with lipoprotein particles or cell surface receptors, may directly mediate this survival benefit. Alternatively, the effect of the APOE4 allele on CLL may be indirect, through its regulation of inflammatory responses, lipoprotein particle distribution, cholesterol metabolism or fatty acid metabolism. Whether the enhanced survival of APOE4 women is due to differences intrinsic to the B-lymphocyte or due to differences in the blood and tissue environments in which these cells reside is also not known.
The frequency of the APOE alleles in the CLL patient population was identical to that of control populations. APOE genotype therefore does not appear to affect susceptibility to CLL, but influences the clinical course of disease, particularly after therapy is initiated. In contrast, APOE genotype does influence susceptibility to other diseases, most notably Alzheimer's, in which APOE4 markedly increases risk.9 The beneficial impact of APOE4 in CLL and its deleterious impact on Alzheimer's disease expression may relate to a common mechanism of apoE4 in enhancing apoptotic cell death.
Chiorazzi N, Rai KR, Ferrarini M . Chronic lymphocytic leukemia. N Engl J Med 2005; 352: 804–815.
Keating MJ . Chronic lymphocytic leukemia. In: Henderson ES, Lister TA, Greaves MF (eds). Leukemia, 7th edn. Saunders: Philadelphia, 2002, pp 131–151.
Cuni S, Perez-Aciego P, Perez-Chacon G, Vargas JA, Sanchez A, Martin-Saavedra FM et al. A sustained activation of PI3K/NF-kappaB pathway is critical for the survival of chronic lymphocytic leukemia B cells. Leukemia 2004; 18: 1391–1400.
DeKroon R, Robinette JB, Hjelmeland AB, Wiggins E, Blackwell M, Mihovilovic M et al. APOE4-VLDL inhibits the HDL-activated phosphatidylinositol 3-kinase/Akt Pathway via the phosphoinositol phosphatase SHIP2. Circ Res 2006; 99: 829–836.
DeKroon RM, Mihovilovic M, Goodger ZV, Robinette JB, Sullivan PM, Saunders AM et al. ApoE genotype-specific inhibition of apoptosis. J Lipid Res 2003; 44: 1566–1573.
Weinberg JB, Volkheimer AD, Chen Y, Beasley BE, Jiang N, Lanasa MC et al. Clinical and molecular predictors of disease severity and survival in chronic lymphocytic leukemia. Am J Hematol 2007; 82: 1063–1070.
Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O′Brien S et al. National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood 1996; 87: 4990–4997.
Volkheimer AD, Weinberg JB, Beasley BE, Whitesides JF, Gockerman JP, Moore JO et al. Progressive immunoglobulin gene mutations in chronic lymphocytic leukemia: evidence for antigen-driven intraclonal diversification. Blood 2007; 109: 1559–1567.
Saunders AM, Strittmatter WJ, Schmechel D, George-Hyslop PH, Pericak-Vance MA, Joo SH et al. Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer’s disease. Neurology 1993; 43: 1467–1472.
Mihovilovic M, Hulette C, Gilbert JR . Strategy to analyze thymic transcription of alpha-3 neuronal nicotinic acetylcholine receptor submit. Methods Neurosc 1993; 12: 169–190.
Davignon J, Cohn JS, Mabile L, Bernier L . Apolipoprotein E and atherosclerosis: insight from animal and human studies. Clin Chim Acta 1999; 286: 115–143.
Schaefer EJ, Lamon-Fava S, Johnson S, Ordovas JM, Schaefer MM, Castelli WP et al. Effects of gender and menopausal status on the association of apolipoprotein E phenotype with plasma lipoprotein levels. Results from the Framingham Offspring Study. Arterioscler Thromb 1994; 14: 1105–1113.
Oppezzo P, Vasconcelos Y, Settegrana C, Jeannel D, Vuillier F, Legarff-Tavernier M et al. The LPL/ADAM29 expression ratio is a novel prognosis indicator in chronic lymphocytic leukemia. Blood 2005; 106: 650–657.
Nuckel H, Huttmann A, Klein-Hitpass L, Schroers R, Fuhrer A, Sellmann L et al. Lipoprotein lipase expression is a novel prognostic factor in B-cell chronic lymphocytic leukemia. Leuk Lymphoma 2006; 47: 1053–1061.
Heintel D, Kienle D, Shehata M, Krober A, Kroemer E, Schwarzinger I et al. High expression of lipoprotein lipase in poor risk B-cell chronic lymphocytic leukemia. Leukemia 2005; 19: 1216–1223.
Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999; 94: 1840–1847.
Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK . Unmutated Ig V-H genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999; 94: 1848–1854.
Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, Bullinger L et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 2000; 343: 1910–1916.
Schwarz JK, Yarbro JW . Scientific basis of cancer chemotherapy. In: Perry MC (ed). The Chemotherapy Source Book. Lippincott Williams & Wilkins: Philadelphia, 2001, pp 1–6.
Ji ZS, Pitas RE, Mahley RW . Differential cellular accumulation/retention of apolipoprotein E mediated by cell surface heparan sulfate proteoglycans. Apolipoproteins E3 and E2 greater than e4. J Biol Chem 1998; 273: 13452–13460.
Mulder M, Lombardi P, Jansen H, van Berkel TJ, Frants RR, Havekes LM . Low density lipoprotein receptor internalizes low density and very low density lipoproteins that are bound to heparan sulfate proteoglycans via lipoprotein lipase. J Biol Chem 1993; 268: 9369–9375.
Molica S . Sex differences in incidence and outcome of chronic lymphocytic leukemia patients. Leuk Lymphoma 2006; 47: 1477–1480.
Huang P, Feng L, Oldham EA, Keating MJ, Plunkett W . Superoxide dismutase as a target for the selective killing of cancer cells. Nature 2000; 407: 390–395.
Narasimhan P, Amaral L . Lymphopenic response of patients presenting with chronic lymphocytic leukemia associated with carcinoma of the prostate to diethylstilbestrol: correlation of response to the in vitro synthesis of RNA by patient lymphocytes and its relationship to transcortin. Am J Hematol 1980; 8: 369–375.
Gao N, Rahmani M, Dent P, Grant S . 2-Methoxyestradiol-induced apoptosis in human leukemia cells proceeds through a reactive oxygen species and Akt-dependent process. Oncogene 2005; 24: 3797–3809.
Haynes MP, Sinha D, Russell KS, Collinge M, Fulton D, Morales-Ruiz M et al. Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells. Circ Res 2000; 87: 677–682.
Wang JM, Irwin RW, Brinton RD . Activation of estrogen receptor alpha increases and estrogen receptor beta decreases apolipoprotein E expression in hippocampus in vitro and in vivo. Proc Natl Acad Sci USA 2006; 103: 16983–16988.
Raber J, Bongers G, LeFevour A, Buttini M, Mucke L . Androgens protect against apolipoprotein E4-induced cognitive deficits. J Neurosci 2002; 22: 5204–5209.
Dallongeville J, Lussier-Cacan S, Davignon J . Modulation of plasma triglyceride levels by apoE phenotype: a meta-analysis. J Lipid Res 1992; 33: 447–454.
Ferrieres J, Sing CF, Roy M, Davignon J, Lussier-Cacan S . Apolipoprotein E polymorphism and heterozygous familial hypercholesterolemia. Sex-specific effects. Arterioscler Thromb 1994; 14: 1553–1560.
We thank the patients for their participation, and the Hematology–Oncology nurses and physician assistants for their special contributions. The work was supported in part by the National Institutes of Health, the Leukemia and Lymphoma Society and the VA Research Service (JBW); Glenn/AFAR (RD); and GlaxoSmithKline and the Deane Laboratory (WJS).
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Weinberg, J., Volkheimer, A., Mihovilovic, M. et al. Apolipoprotein E genotype as a determinant of survival in chronic lymphocytic leukemia. Leukemia 22, 2184–2192 (2008). https://doi.org/10.1038/leu.2008.241
- lipoprotein lipase
- chronic lymphocytic leukemia
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Cancer Epidemiology Biomarkers & Prevention (2010)
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Reduced expression of the tumor suppressor PHLPP1 enhances the antiapoptotic B-cell receptor signal in chronic lymphocytic leukemia B-cells