Gerard Tobin

Association between telomere length and V H gene mutation status in chronic lymphocytic leukaemia: clinical and biological implications

The immunoglobulin VH gene mutation status can divide B-cell chronic lymphocytic leukaemia (CLL) into two entities with a different clinical course. Cases with unmutated VH genes, considered to evolve from pregerminal centre (GC) cells, have a worse outcome compared to cases showing mutated VH genes, that is, post-GC derived. Also, telomere length has been reported to be of prognostic significance in CLL. Interestingly, telomerase becomes activated during the GC reaction and an elongation of the telomeres occurs in GC B cells. We performed telomere length and VH gene analysis in a series of 61 CLL cases, in order to investigate if the unique telomere lengthening shown in GC B cells could reflect the telomere status in the two subsets of mutated and unmutated CLL. A novel association was found between VH gene mutation status and telomere length, since significantly shorter telomeres were demonstrated in the unmutated group compared to the mutated group (mean length 4.3 vs 6.3 kbp). Shorter telomeres also constituted a subgroup with a worse prognosis than cases with longer telomeres (median survival 59 vs 159 months). Furthermore, the Ig gene sequence data revealed that samples with high mutations frequency (>6%) had long telomeres (∼8 kbp). Thus, both the telomere and VH gene mutation status in CLL appear linked, which may reflect the proliferative history of the clonal cells with regard to the GC reaction.

Upregulation of bfl-1 is a potential mechanism of chemoresistance in B-cell chronic lymphocytic leukaemia

B-cell chronic lymphocytic leukaemia (B-CLL) is characterised by the progressive accumulation of monoclonal CD5+ B cells. In a previous study, we have analysed the expression profile of apoptosis-regulating genes using a cDNA-based microarray and found overexpression of the antiapoptotic bcl-2 family member, bfl-1, in B-CLL cells with an apoptosis-resistant phenotype. In this study, bfl-1 mRNA levels have been determined by competitive PCR in an extended population of B-CLL patients to characterise its role in disease progression and development of chemoresistance. bfl-1 levels were significantly higher in patients with no response (NR) to last chemotherapy than in patients responding (partial response (PR)) to last chemotherapy (P<0.05) and in patients who had not required treatment (P<0.05). We found no correlation between bfl-1 mRNA levels and disease progression, IGHV mutational status or other clinical parameters. In addition, bfl-1 mRNA levels were inversely correlated with apoptotic response to in vitro fludarabine treatment of B-CLL cells. Specific downregulation of bfl-1 using siRNA induced apoptosis in resistant cells. Our data suggest that bfl-1 contributes to chemoresistance and might be a therapeutic target in B-CLL.

VH3-21 gene usage in chronic lymphocytic leukemia: Characterization of a new subgroup with distinct molecular features and poor survival

During recent years it has become evident that lymphoproliferative diseases of B-cell origin display preferential immunoglobulin (Ig) variable heavy chain (VH) gene usage. For instance, the VH1-69 and VH4-34 genes were early found to be overexpressed in B-cell chronic lymphocytic leukemia (CLL) and other B-cell lymphomas. The implications of biased VH gene usage have been speculated to be a result of stimulation of unknown antigens, which gives increased proliferation of B-cells with certain VH gene configuration and consequently higher probability to undergo transformation. Thus, VH gene usage may play a role in development of leukemias and lymphomas. Recently, we could confirm the over usage of the VH1-69 and VH4-34 genes in CLL, but a novel finding was that the VH3-21 gene was preferentially utilized in CLL patients with mutated VH genes. These VH3-21+ Ig rearrangements showed molecular peculiarities such as shorter lengths of the third complementarity determining region (CDR) and had similar amino acid composition of their CDR3s, implicating recognition of the same antigen in individual tumors. Most of the VH3-21+ patients also showed a predominance of λ chain expression and biased usage of 1 specific Vλ gene, V2-14. Furthermore, overall survival appeared to correlate with VH3-21 usage and, regardless of VH gene mutation status, VH3-21+ patients had a poor outcome. All in all, it appears that VH3-21 gene usage define a new entity of CLL. The remaining question now to be clarified is if antigen(s) actually are involved in the pathogenesis of VH3-21+ CLL.

Association of the 1513C polymorphism in the P2X7 gene with familial forms of chronic lymphocytic leukaemia

Association of the 1513C polymorphism in the P2X7 gene with familial forms of chronic lymphocytic leukaemia.

The G(-248)A polymorphism in the promoter region of the Bax gene does not correlate with prognostic markers or overall survival in chronic lymphocytic leukemia

The G(-248)A polymorphism in the promoter region of the Bax gene was recently associated with low Bax expression, more advanced stage, treatment resistance and short overall survival in B-cell chronic lymphocytic leukemia (CLL), the latter particularly in treated patients. To investigate this further, we analyzed 463 CLL patients regarding the presence or absence of the G(-248)A polymorphism and correlated with overall survival, treatment status and known prognostic factors, for example, Binet stage, VH mutation status and genomic aberrations. In this material, similar allele and genotype frequencies of the Bax polymorphism were demonstrated in CLL patients and controls (n=207), where 19 and 21% carried this polymorphism, respectively, and no skewed distribution of the polymorphism was evident between different Binet stages and VH mutated and unmutated CLLs. Furthermore, no difference in overall survival was shown between patients displaying the G(-248)A polymorphism or not (median survival 85 and 102 months, respectively, P=0.21), and the polymorphism did not influence outcome specifically in treated CLL. Neither did the polymorphism affect outcome in prognostic subsets defined by VH mutation status or genomic aberrations. In conclusion, the pathogenic role and clinical impact of the Bax polymorphism is limited in CLL.

Lipoprotein lipase is differentially expressed in prognostic subsets of chronic lymphocytic leukemia but displays invariably low catalytical activity

Lipoprotein lipase (LPL) expression has been shown to correlate with IGHV mutational status and to predict outcome in chronic lymphocytic leukemia (CLL). We here investigated the prognostic impact of LPL expression in relation to other prognostic markers including IGHV3-21 usage in 140 CLL patients. Additionally, we studied the catalytic activity of LPL in CLL cells. A significant difference in LPL mRNA expression was detected in IGHV unmutated compared to mutated CLL patients (p<0.001). However, the poor-prognostic mutated/stereotyped IGHV3-21 patients did not differ from other mutated CLL cases. Clinical outcome was significantly different in CLL cases with high versus low LPL expression (p<0.001), and LPL expression exceeded mutation status/IGHV3-21 usage as an independent prognostic marker. Finally, LPL protein expression correlated significantly with mRNA expression and was higher in IGHV unmutated versus mutated CLL (p=0.018), although the majority of synthesized protein was catalytically inactive indicating a non-catalytical function in CLL.

Thioredoxin, produced by stromal cells retrieved from the lymph node microenvironment, rescues chronic lymphocytic leukemia cells from apoptosis in vitro

Background and Objectives The redox-regulatory protein thioredoxin has several functions including transcriptional regulation, and antioxidant, cytokine, and chemokine activities. We have previously shown that extracellular thioredoxin protects B-cell chronic lymphocytic leukemia (CLL) cells from apoptosis in vitro. In this study we were interested to determine whether thioredoxin is produced by cells surrounding the CLL cells in the in vivo microenvironment and whether this cell-derived thioredoxin has any leukemia growth-promoting effect in vitro. Design and Methods Lymph nodes from CLL patients (n=25) were analyzed for thioredoxin expression by immunohistology. Stromal cells purified from the lymph nodes were analyzed for thioredoxin secretion at the single cell level using an ELIspot assay. The survival effect of the stromal-derived thioredoxin was tested by co-culturing stromal- and CLL cells with and without Fab-fragments of an anti-thioredoxin antibody. Results The results indicated that the thioredoxin production correlated with the amount of proliferating cells and was mainly localized to the proliferation centers (pseudofollicles) in the CLL lymph nodes. The leukemia cells per se showed minimal thioredoxin levels; in contrast, stromal cells strongly expressed thioredoxin. Purified primary stromal cells, which secreted extracellular thioredoxin, significantly protected the CLL cells from undergoing apoptosis in 72 h co-cultures. Interestingly, this anti-apoptotic effect could be abrogated by addition of Fab-fragments of an anti- thioredoxin antibody. Interpretation and Conclusions In conclusion, we have shown that stromal cells in the lymph node microenvironment produce thioredoxin and that the thioredoxin production is localized to the proliferation centers of the CLL lymph nodes. In addition, thioredoxin produced by purified stromal cells rescued CLL cells from apoptosis in vitro.

Prognostic usage of V(H) gene mutation status and its surrogate markers and the role of antigen selection in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease with many patients surviving for decades with minimal or no treatment, whereas others succumb rapidly to their disease despite therapy. In recent years, new molecular prognostic factors have emerged in CLL that have significantly improved the subgrouping of the disease. One of the most important molecular predictors, the immunoglobulin VH gene mutation status, divides CLL into two prognostic groups, depending on the presence or absence of somatic hypermutation, where unmutated VH genes are associated with considerably worse prognosis than mutated VH genes. An exception to this appears to be CLL patients utilizing the VH3-21 gene as they have poor outcome irrespective of mutation status. Surrogate markers for the VH gene mutation status have been suggested, such as CD38 and ZAP-70 expression. However, the CD38 level was later shown to display poor correlation to the mutation status, although it may still serve as an independent prognostic factor. More promising is the expression levels of ZAP-70, which appears to be both a strong surrogate marker for VH gene mutation status, although discrepancies have been reported, as well as an independent prognostic marker. Immunoglobulin gene analysis has also indicated the possibility of antigen selection in CLL considering the significant bias in VH gene usage. Intriguingly, the VH3-21+ group and several other CLL subsets using certain VH genes was recently reported to display strikingly restricted immunoglobulin gene features, in both their heavy and light chain gene rearrangements, thus further high-lighting the possible role of antigen involvement in CLL development.

Surface antigen expression and correlation with variable heavy-chain gene mutation status in chronic lymphocytic leukemia

Abstract: Recent studies have demonstrated that B‐cell chronic lymphocytic leukemia (CLL) consists of two clinical entities with either somatically hypermutated (M‐CLL) or unmutated (UM‐CLL) immunoglobulin variable heavy‐chain (VH) regions. In view of the fact that the cellular biology of these two subsets of disease is currently unexplored, we performed an extensive analysis of the surface antigen expression and correlated this with the VH gene mutation status in a cohort of 32 CLL patients. Using polymerase chain reaction amplification and nucleotide sequencing, the VH genes were shown to be mutated in 10 cases (31%) and unmutated in 22 (69%). The expression of 27 surface membrane antigens in peripheral blood leukemic cells was analyzed by flow cytometry, measuring both the percentage of positive cells as well as the geometric mean fluorescence intensity (GMF). Most of the surface membrane antigens (CD5, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD45, VD79b, CD80, CD95, CD122, CD124, CD126, CD130, CD154, IgM, and IgD) showed a similar expression pattern in both UM‐CLL and M‐CLL patients. The similarity of M‐CLL and UM‐CLL, as demonstrated here for the first time with many protein markers, indicates a considerably homogenous phenotype in both subsets. Furthermore, CD27 was strongly expressed in all cases, which may suggest a memory cell phenotype for both M‐CLL and UM‐CLL. More positive cells in the UM‐CLL group were observed regarding CD38, but CD38 was not a good predictor of VH gene mutation status. Seventy percent of the M‐CLL cases, but only 36% of UM‐CLL cases, were Ig‐λ+. The most striking differential expression, however, was observed in the two slicing variants of the common leukocyte antigen CD45, namely CD45RO and CD45RA. CD45RO expression was significantly associated with M‐CLL, whereas the GMF intensity of CD45RA tended to be associated with UM‐CLL. The role of these CD45 splicing variants in the pathogenesis of CLL deserves further investigation.

Rapamycin shows anticancer activity in primary chronic lymphocytic leukemia cells in vitro, as single agent and in drug combination.

The mammalian target of rapamycin inhibitor rapamycin and its analogues show promising anticancer activity in various experimental tumor models and are presently evaluated in clinical trials. We, here, evaluated the in vitro activity of rapamycin with regard to tumor-type specificity and possible mechanisms of drug resistance in 97 tumor cell samples from patients and in a resistance-based cell line panel, using the fluorometric microculture cytotoxicity assay. Rapamycin was dose-dependently cytotoxic in patient tumor cells and in cell lines. In primary cells, rapamycin was more active in hematological than in solid tumor samples, with chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia being the most sensitive tumor types. Considerable inter-individual differences in sensitivity were apparent among CLL samples, but no difference was observed between IGHV mutated and unmutated CLL samples, whereas a tendency to lower rapamycin sensitivity was indicated for samples displaying poor-prognostic genomic markers. Combination experiments in CLL cells indicated that rapamycin acted synergistically with vincristine, cisplatin, chlorambucil and taxotere. These results and the clinically-experienced good tolerance to rapamycin analogues encourage clinical studies of rapamycin in CLL treatment as single agent but also in combination with, e.g., vincristine and chlorambucil.