Karen M. Bond

A Genomic Approach to Improve Prognosis and Predict Therapeutic Response in Chronic Lymphocytic Leukemia

Purpose: Chronic lymphocytic leukemia (CLL) is a B-cell malignancy characterized by a variable clinical course. Several parameters have prognostic capabilities but are associated with altered response to therapy in only a small subset of patients. Experimental Design: We used gene expression profiling methods to generate predictors of therapy response and prognosis. Genomic signatures that reflect progressive disease and responses to chemotherapy or chemoimmunotherapy were created using cancer cell lines and patient leukemia cell samples. We validated and applied these three signatures to independent clinical data from four cohorts, representing a total of 301 CLL patients. Results: A genomic signature of prognosis created from patient leukemic cell gene expression data coupled with clinical parameters significantly differentiated patients with stable disease from those with progressive disease in the training data set. The progression signature was validated in two independent data sets, showing a capacity to accurately identify patients at risk for progressive disease. In addition, genomic signatures that predict response to chlorambucil or pentostatin, cyclophosphamide, and rituximab were generated and could accurately distinguish responding and nonresponding CLL patients. Conclusions: Thus, microarray analysis of CLL lymphocytes can be used to refine prognosis and predict response to different therapies. These results have implications for standard and investigational therapeutics in CLL patients. (Clin Cancer Res 2009;15(22):694755)

Apolipoprotein E genotype as a determinant of survival in chronic lymphocytic leukemia

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.

LMP-420: a novel purine nucleoside analog with potent cytotoxic effects for CLL cells and minimal toxicity for normal hematopoietic cells

B-cell chronic lymphocytic leukemia (CLL) is characterized by slow accumulation of malignant cells, which are supported in the microenvironment by cell–cell interactions and soluble cytokines such as tumor necrosis factor (TNF). We evaluated the effect of the small molecule TNF inhibitor LMP-420 on primary CLL cells. The mean concentration of LMP-420 required to induce 50% cytotoxicity (ED50) at 72 h was 245 n. LMP-420-induced time- and dose-dependent apoptosis, as shown by annexin V staining, caspase activation and DNA fragmentation. These changes were associated with decreased expression of anti-apoptotic proteins Mcl-1, Bcl-xL and Bcl-2. CLL cells from patients with poor prognostic indicators showed LMP-420 sensitivity equal to that for cells from patients with favorable characteristics. In addition, LMP-420 potentiated the cytotoxic effect of fludarabine and inhibited in vitro proliferation of stimulated CLL cells. Gene expression profiling indicated that the mechanism of action of LMP-420 may involve suppression of nuclear factor-κB and immune response pathways in CLL cells. LMP-420 had minimal effects on normal peripheral blood mononuclear cell, B- and T-cell function, and hematopoietic colony formation. Our data suggest that LMP-420 may be a useful treatment for CLL with negligible hematologic toxicities.

Oligoclonal TRBV gene usage among CD8+ T cells in monoclonal B lymphocytosis and CLL

Whereas most malignancies are associated with decreased numbers of circulating T cells, in chronic lymphocytic leukemia (CLL) T cell numbers are elevated 2 to 4 times normal. Though T cells are increased in number, the T cell repertoire is significantly contracted in CLL, with oligoclonal and monoclonal subsets (Goolsby, et al 2000, Rezvany, et al 1999). Rather than promoting an anti-tumor response, these qualitative and quantitative T cell abnormalities may contribute to a tumor microenvironment that promotes progression of the malignant clone (Ghia and Caligaris-Cappio 2000). CLL patients with mutated immunoglobin heavy chain variable region (IgVH) genes follow a more indolent disease course than those with unmutated IgVH. It has been proposed that IgVH unmutated CLL arise from B cells that mature through a T cell independent pathway, because in the germinal center reaction CD4+ T cell help is required for somatic mutation and isotype switching. Therefore, IgVH mutated and unmutated CLL may have differing capacities for T cell interaction and immune modulation. A recent report by Palmer et al showed that increased numbers of T and “natural killer” (NK) cells relative to the number of CLL lymphocytes in patients with newly diagnosed CLL was associated with both a mutated IgVH and improved prognosis, suggesting that T and NK cell responses precede clinical CLL (Palmer, et al 2008). Using sensitive flow cytometry, we and other groups have found that small populations of cells with a typical CLL immunophenotype can be found in older adults (Ghia, et al 2004, Rawstron, et al 2002). This condition is termed monoclonal B-lymphocytosis (MBL). Because MBL may progress to CLL (Rawstron, et al 2008), we sought to clarify if either the number or T cell receptor (TCR) repertoire of T cells differed between IgVH mutated CLL, and IgVH unmutated CLL, and MBL. Twenty untreated CLL patients were identified from a longitudinal CLL patient cohort at Duke University or the Durham VA Medical Centers between October 2006 and August 2007. The clinical and molecular characteristics of this longitudinal cohort of patients have been previously described (Weinberg, et al 2007). A priori, an equal number of IgVH mutated and unmutated patients were selected for this study. No other clinical or molecular features were used to identify or stratify patients. A diagnosis of CLL was based on standard criteria (Weinberg, et al 2007). MBL subjects were identified by flow cytometric screening of unaffected relatives of patients with familial CLL at Duke University or the Durham VA Medical Center. Eight subjects from 5 different families with MBL provided blood for T cell analysis. This study was approved by the Institutional Review Boards at Duke University and the Durham VA Medical Centers, and all subjects provided written informed consent. CLL and MBL subject characteristics are summarized in Table 1. Patients with IgVH unmutated and mutated CLL were similar in regards to WBC count, age, lymphocyte doubling time, and duration since diagnosis. Similar to prior reports, unmutated IgVH correlated with ZAP70 expression (Fishers exact test, p<0.01) and surface expression of CD38 (Fishers exact test, p<0.05). Patients with IgVH unmutated CLL were significantly more likely to be intermediate or high Rai Stage (χ2df=2, p<0.01). The mean age of CLL patients was 62, and the mean MBL age was 71. This difference was not statistically significant (2-sided t-test, p=0.1). Table 1 Subject Characteristics Lymphocyte subset enumeration was performed using multi-channel flow cytometry (Table 2 and Supporting Information). The absolute B cell count was not significantly different in IgVH unmutated compared to mutated CLL patients. Among MBL subjects, the mean absolute B cell count was 0.6 ×109 cells/L and the MBL clone size was small (< 150 ×106 cells/L) in 7 of the 8 subjects. The CD4+ count was similar in IgVH unmutated CLL, IgVH mutated CLL and MBL subjects. The CD8+ count was higher in IgVH unmutated compared to IgVH mutated CLL patients (891 vs. 593 ×106 cells/L, n.s.), and was significantly higher in IgVH unmutated than MBL subjects (891 vs. 273 ×106 cells/L, p<0.05). Similarly, the ratio of CD4+:CD8+ T cells was significantly lower in IgVH unmutated CLL compared to IgVH mutated CLL or MBL (1.6 vs. 2.6, p<0.05, and 1.6 vs. 4.5, p<0.01, respectively). NK T cell numbers were not significantly different among IgVH unmutated CLL compared to IgVH mutated CLL or MBL. Table 2 Lymphocyte Enumeration and T Cell Repertoire Divergence Coefficients To estimate the CD4+ and CD8+ T cell repertoire diversity, we coupled PCR amplification of the CDR3 length polymorphism of 23 TCRVβ families (“spectratyping”) with a novel, quantitative method for analysis of TCR repertoires by using SpA (Kepler, et al 2005) (Supporting information). CD4+ and CD8+ T cells were positively selected using anti-CD4 and anti-CD8 monoclonal antibodies conjugated to magnetic beads according to the manufacturers directions (Dynabeads®, Invitrogen, Carlsbad, CA). The calculated mean divergence coefficients (a calculated unitless value to quantify the degree of “skewness” from a pseudo-Gaussian distribution) for the T cell spectratypes are provided in Table 2. Combined analysis of all CLL patients showed the mean CD8+ TCR spectratype divergence was 0.32, significantly higher than the mean CD4+ divergence (0.07, 2 sided t-test, p<10−6). Oligoclonal and monoclonal Vβ families were frequently observed among CD8+ cells, but rarely observed among CD4+ cells. We did not observe restriction of any specific Vβ families across patients. Given our hypothesis that IgVH unmutated CLL and mutated CLL may have different capacities for T cell interaction, we compared the TCR repertoire between these patient groups. The mean CD8+ TCR spectratype divergence was greater in IgVH unmutated CLL than in mutated CLL among CD8+ cells, but this difference was not statistically significant (0.36 vs. 0.29, 2-sided t-test, p=0.08, n.s). Next, we evaluated whether MBL have a restricted TCR repertoire. The mean CD8+ and CD4+ divergence among MBL subjects was similar to that observed in CLL patients (mean CD8+ divergence: 0.35 for MBL vs. 0.32 for all CLL; mean CD4+ divergence: 0.06 for MBL vs. 0.07 for all CLL, Table 2). We performed a multivariate correlation to identify variables associated with restriction of the CD8+ repertoire in MBL. Patient characteristics, the absolute number of different lymphocyte subsets, and “MBL characteristics” (ZAP70 status, CD38 status, and the “MBL percentage,” defined as the number of MBL cells divided by the total number of CD19+ B cells) were considered. A correlation between the absolute number of CD4+ cells and the diversity of the CD8+ repertoire was observed: higher numbers of CD4+ cells correlated with more diverse CD8+ repertoire (ρ = −0.82, p<0.05). Loss of specific CD4+ subsets may cause contraction of the CD8+ repertoire; alternatively some CLL and MBL may have the capacity to modulate both the CD4+ and CD8+ subsets as independent effects. Of the remaining variables, a correlation was observed between the MBL percentage and mean CD8+ divergence (ρ = 0.52, n.s.). There was no correlation of mean CD8+ divergence with MBL subject age (ρ = 0.20, n.s.). Because abnormalities in the T cell compartment are detectable at time of CLL diagnosis (Palmer, et al 2008), we speculate that MBL lymphocytes may have a capacity for T cell immune modulation similar to CLL lymphocytes. The absolute number of MBL lymphocytes was quite small in this cohort of MBL subjects, suggesting that acquisition of an immunomodulatory phenotype is an early event in CLL leukemogenesis. Further investigation is required to test this hypothesis. Furthermore, MBL is relatively common among the elderly (Ghia, et al 2004), and the great majority of MBL likely do not progress to CLL (Rawstron, et al 2008). We identified qualitative abnormalities in the T cell repertoire in this otherwise healthy group of elderly individuals, suggesting that MBL may contribute to accelerated immune senescence.