Angiopoietin-2 expression in B-cell chronic lymphocytic leukemia: association with clinical outcome and immunoglobulin heavy-chain mutational status

Abstract B-cell Chronic Lymphocytic Leukemia (B-CLL) follows an extremely variable clinical course. For some patients CLL is an indolent disease that never progresses to the point of requiring therapy and these patients have a survival time similar to age-matched controls. On the contrary, other patients experience rapidly deteriorating blood count and organomegaly which requires prompt treatment. The overall survival (OS) times range from months to decades. B-CLL patients can be divided into two subgroups on the basis of the presence or absence of somatic mutations in the specific immunoglobulin heavy-chain variable region (IgVH) genes used by leukemic cells. Patients with unmutated IgVH genes usually have an advanced stage and an unfavourable cytogenetic features, require therapy and have a short survival. The biological reasons of the different behaviour of Ig-mutated and Ig-unmutated leukemic clones have not been fully elucidated yet. Angiogenesis is a very complex network which is closely regulated by the orchestrating functions of many angiogenic factors. The balance of cellular expression of all these angiogenic factors determines vascular stabilization or angiogenic remodelling and sprouting or vessel regression. Increasing evidence shows that neovascularization plays a role in the biology of chronic lymphocytic leukemia. The goal of this study was to evaluate the angiogenic status of Ig-mutated and Ig-unmutated CLL in the attempt to identify a possible role of angiogenesis in the adverse clinical outcome of Ig-unmutated CLL patients. So, we first performed a large scale gene-expression analysis on 29 B-CLL patients using microarrays comprising about 20,000 probes and 208 angiogenesis-related genes. We identified 64 up-regulated genes in Ig-unmutated CLL relative to Ig-mutated CLL. Among them, we found angiopoietin-2 (Ang-2) as one of the highest differentially expressed gene (p=3.02x10−6). Then, we evaluated the Ang-2 expression both at transcript and protein level in a wide cohort of CLL, in normal controls and in other haematological malignancies. The data showed an extremely wide range of Ang-2 expression in B-CLL: Ang-2 high-expressing cases were characterized by advanced Binet stage (p=0.032) and significantly shorter progression-free survival than Ang-2 low-expressing subset (median, 16 vs. 146 months) (p=0.006). Moreover, there was a strong correlation between the IgVH mutational status and the Ang-2 gene expression level (p<0.0001). Ig-mutated CLL exhibited very low Ang-2 expression, absolutely similar to the levels measured in healthy controls (median, 0.27 vs. 0.32, p=0.959). On the contrary, Ig-unmutated CLL expressed up to one hundred-fold higher levels of Ang-2 than normal controls(median, 38.61 vs. 0.32, p=0.002). Moreover, Ang-2 was up-regulated in all investigated CML, ALL and AML samples. We observed extremely high levels of expression in CML and ALL (median Ang-2 mRNA, 3948.96 and 190.00, respectively), whereas moderately high levels of Ang-2 were found in AML patients (median, 16.21). These data suggest that increased angiogenesis due to high Ang-2 expression may be involved in adverse clinical outcome of Ig-unmutated CLL patients.

Download Full-text

Serum BAFF (B-Cell Activating Factor of the TNF Family) Compared with Immunoglobulin Heavy-Chain Mutation Status, ZAP-70 and CD38 as a Predictor of Time to First Treatment in Early B-Cell Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v110.11.3093.3093 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3093-3093

Author(s):

Stefano Molica ◽

Gaetano Vitelli ◽

Giovanna Cutrona ◽

Giovanna Digiesi ◽

Rosanna Mirabelli ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Heavy Chain ◽

Immunoglobulin Heavy Chain ◽

Serum Levels ◽

Lymphocytic Leukemia ◽

B Cell Activating Factor ◽

Mutational Status ◽

Time To First Treatment ◽

Cell Chronic Lymphocytic Leukemia

Abstract We analyzed the correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukemia [CLL] (i.e, mutational status of the immunoglobulin heavy chain variable region [IgVH], ZAP-70- and CD38-expression) and serum levels of BAFF (B-cell activating factor of the TNF family) by evaluating the impact of these variables on the time to first treatment [TFT] in a series of 69 previously untreated Binet stage A B-cell CLL patients. By using a commercial ELISA (R & D Systems, USA) we found that higher levels of BAFF characterized more frequently patients with Rai stage 0 (P=0.008) and mutated IgVH (P=0.03). In contrast, peripheral blood lymphocytosis (P=0.06), serum β2-m (P=0.159), LDH (P=0.333) and percentage of ZAP-70-positive (P=0.242) or CD38-positive B-CLL cells (P=0.142) did not reflect circulating levels of BAFF. The relationship among various bio-pathological parameters, analyzed by the multiple correspondence analysis (MCA), showed two different clinico-biological profiles. The first, characterized by higher BAFF serum levels (i.e., > 336 ng/mL), presence of mutation in the IgVH, low percentage of CD38-positive B-CLL cells (< 30%) and low LDH was associated with a stable pattern of disease generally not requiring therapy. The second, defined by lower BAFF levels, absence of mutation in the IgVH, high percentage of CD38- positive B-CLL cells and high LDH was associated with a more progressive pattern of disease and a shorter TFT. After a median follow-up time of 35 months (range, 2–120 months) 26 (37.6%) out of 69 patients experienced a need for chemotherapy. Kaplan-Meier estimates of patientsTFT, plotted after searching the best cut-off for BAFF (i.e., 336 ng/mL), demonstrated that low BAFF concentration was associated with a shorter TFT (median TFT 36 months) while median was not reached by patients with BAFF levels higher than 336 ng/mL (P<0.0001). Along with lower serum levels of BAFF (Hazard Ratio [HR], 0.19; P<0.0001), the univariate Cox proportional hazard model identified absence of mutation in IgVH (HR, 0.17; P<0.0001), CD38-positivity (HR, 3.32; P=0.01) and lower platelet count (HR, 0.19; P=0.03) as predictor of shorter TFT. Finally, in multivariate analysis only mutational status of IgVH (HR, 0.25; P=0.007) and serum concentration of BAFF (HR, 034; P=0.04) affected significantly TFT. Our results indicate that in early B-cell CLL clinico-biological profile including among other parameters BAFF may provide a useful insight into the complex interrelationship of prognostic variables and semplify their interpretation. The possible presence of BAFF isoform in B-CLL could peraphs account for the unexpected correlation between low soluble BAFF levels and poor clinical outcome in patients with early disease.

Download Full-text

Laboratory, Morphologic, and Immunophenotypic Correlates of Surface Immunoglobulin Heavy Chain Isotype Expression in B-Cell Chronic Lymphocytic Leukemia

American Journal of Clinical Pathology ◽

10.1309/1tyf-vpm9-cq2c-b0tf ◽

2001 ◽

Vol 116 (6) ◽

pp. 905-912 ◽

Cited By ~ 6

Author(s):

Peter U.F. Shen ◽

Sheila G. Fuller ◽

William N. Rezuke ◽

Bradford J. Sherburne ◽

Joseph A. DiGiuseppe

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Heavy Chain ◽

Immunoglobulin Heavy Chain ◽

Lymphocytic Leukemia ◽

Surface Immunoglobulin ◽

Cell Chronic Lymphocytic Leukemia ◽

Isotype Expression

Download Full-text

Most immunoglobulin heavy chain switch mu rearrangements in B-cell chronic lymphocytic leukemia are internal deletions

FEBS Letters ◽

10.1016/s0014-5793(02)02672-8 ◽

2002 ◽

Vol 518 (1-3) ◽

pp. 119-123 ◽

Cited By ~ 10

Author(s):

Elena Nardini ◽

Simona Rizzi ◽

Daniela Capello ◽

Umberto Vitolo ◽

Gianluca Gaidano ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Heavy Chain ◽

Immunoglobulin Heavy Chain ◽

Lymphocytic Leukemia ◽

Cell Chronic Lymphocytic Leukemia

Download Full-text

Serum Adiponectin Compared with ZAP-70, CD38 and Immunoglobulin Heavy-Chain Mutation Status as a Predictor of Time to First Treatment in Early B-Cell Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v108.11.2786.2786 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2786-2786

Author(s):

Stefano Molica ◽

Gaetano Vitelli ◽

Giovanna Cutrona ◽

Giovanna Digiesi ◽

Rosanna Mirabelli ◽

...

Keyword(s):

Platelet Count ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Heavy Chain ◽

Serum Levels ◽

Lymphocytic Leukemia ◽

Mutational Status ◽

Igvh Mutational Status ◽

Time To First Treatment ◽

Cell Chronic Lymphocytic Leukemia

Abstract We analyzed the correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukemia [CLL] (i.e, mutational status of the immunoglobulin heavy chain variable region [IgVH], ZAP-70- and CD38-expression) and serum levels of adiponectin by evaluating the impact of these variables on the time to first treatment [TFT] in a series of 69 previously untreated Binet stage A B-cell CLL patients. Higher levels of adiponectin inversely correlated with peripheral blood lymphocytosis (r=−0.254; P=0.03), male gender (P=0.0002), percentage of ZAP−70-positive (r=− 0.285; P=0.04) and CD38-positive (r=−0.294; P=0.04) B-CLL cells. After a median follow-up time of 32 months (range, 2–120 months) 28 (40.5%) out of 69 patients experienced a need for chemotherapy. Kaplan-Meier estimates of patients’ TFT, plotted after searching the best cut-off for adiponectin (i.e., 5.88 μg/mL ), demonstrated that low adiponectin concentration was associated with a shorter TFT (median TFT 32 months; P=0.01). The relationship among the various bio-pathological parameters, analyzed by the multiple correspondence analysis (MCA), showed two different clinico-biological profiles. The first, characterized by higher adiponectin serum levels (i.e., > 5.88 μg/mL), higher platelet count (> 174 x 109/L), lower β2-microglobulin [β2-m] (< 2.35 mg/L), presence of mutation in the IgVH and low percentage of either CD38-positive (< 20%) or ZAP-70-positive (< 20%) B-CLL cells, was associated with a stable pattern of disease generally not requiring therapy. The second, defined by lower adiponectin levels, lower platelet count, high β2-m concentration, absence of mutation in the IgVH, high percentage of CD38- and ZAP-70 positive B-cells, was associated with a more progressive pattern of disease and a shorter TFT. The univariate Cox proportional hazard model demonstrated that in addition with lower serum levels of adiponectin (Hazard Ratio [HR], 2.936; P=0.01), the absence of IgVH mutational status (HR, 6.378; P=0.002) and ZAP-70-positivity (HR, 3.314; P=0.02) were associated with a shorter TFT. However, in multivariate analysis only ZAP-70 positivity emerged as predictor of the TFT (HR, 5.187; P=0.008). Our results indicate that in early B-cell CLL clinico-biological profile including among other parameters adiponectin may provide a useful insight into the complex interrelationship of prognostic variables and semplify their interpretation. However, adiponectin may not replace the need for the determination of ZAP-70 and IgVH mutational status.

Download Full-text

Relation of Gene Expression Phenotype to Immunoglobulin Mutation Genotype in B Cell Chronic Lymphocytic Leukemia

Journal of Experimental Medicine ◽

10.1084/jem.194.11.1639 ◽

2001 ◽

Vol 194 (11) ◽

pp. 1639-1648 ◽

Cited By ~ 766

Author(s):

Andreas Rosenwald ◽

Ash A. Alizadeh ◽

George Widhopf ◽

Richard Simon ◽

R. Eric Davis ◽

...

Keyword(s):

Gene Expression ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Germ Line ◽

Gene Expression Signature ◽

Lymphocytic Leukemia ◽

Common Mechanism ◽

Human Leukemia ◽

Mutational Status ◽

Cell Chronic Lymphocytic Leukemia

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression “signature,” irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

Download Full-text

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.

Download Full-text

Analysis of Stereotyped IGHV Distribution In a Series of 1133 Chronic Lymphocytic Leukemia Patients: The Experience of a Multicenter Italian Study Group

Blood ◽

10.1182/blood.v116.21.2423.2423 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2423-2423

Author(s):

Francesco Maura ◽

Giovanna Cutrona ◽

Massimo Gentile ◽

Serena Matis ◽

Monica Colombo ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Heavy Chain ◽

Conflicts Of Interest ◽

Pairwise Alignment ◽

Immunoglobulin Heavy Chain ◽

Lymphocytic Leukemia ◽

Study Group ◽

Data Set ◽

Italian Study ◽

Mutational Status

Abstract Abstract 2423 Chronic lymphocytic leukemia (CLL) is characterized by an extremely variable clinical course. Mutational status of the immunoglobulin heavy-chain variable (IGHV) region defines two disease subsets with different prognosis. A fraction of CLL cases carries highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3). We performed sequence analysis to characterize IGHV regions in a panel of 1133 CLL patients investigated by a multicenter Italian study group. A total of 1148 rearrangements were identified; the analysis of stereotyped subsets was performed based on previously reported criteria (Messmer et al, J Exp Med 2004; Stamatopuolos et al, Blood 2007). Specifically, we compared all our sequences with those found in three different publicly available data sets (Stamatopoulos et al, Blood 2007; Murray et al, Blood 2008 and Rossi et al, 2009 Clin Cancer Res). In addition, a pairwise alignment within all sequences was performed in order to discover novel potential subsets (HCDR3 identity > 60%). Based on the 2% cut-off used to discriminate between Mutated (M) and Unmutated (UM) cases, 777 sequences (67.59%) were classified as M, while 371 sequences (32.3%) as UM. The most represented IGHV genes within mutated cases were IGHV4-34 (104/118) and IGHV3-23 (85/96), whereas IGHV1-69 (97/112) was the most frequently used in the UM group. Interestingly, the IGHV3-21 gene, reported to be frequently expressed in CLL patients from Northern Europe, was present in only a small fraction of cases (24; 2.07%), confirming a previous finding reported by Ghia et al (Blood 2005) in a smaller panel. In our series, stereotyped HCDR3 sequences were found in 407/1148 (35.45%) patients, 177 of whom were M and 230 were UM cases. Overall, we observed that stereotyped sequences were significantly associated with UM IGHV status (Fisher's exact test, P<0.0001). Among the 407 stereotyped HCDR3 sequences, 345 belong to the clusters reported by Murray et al and 14 to those described by Rossi et al., 2009 Clin Cancer Res. The most frequent stereotyped subsets identified in our panel were #1 (35 cases), #7 (28 cases), #4 (24 cases), #3 and #9 (16 cases), #28 (13 cases), and #2 (12 cases), together with subsets #5, #8, #10, #12, #13, #16 and #22 (all ranging from 6 to 9 cases). Finally, we were able to identify by auto-matching analysis 48 sequences potentially specific for 23 novel putative stereotype subsets. In our series we identified 407/1148 (35.45%) stereotyped HCDR3 sequences. The percentage was higher than that reported by Stamatopoulos et al and Murray et al. This discrepancy may partially be due to the different approach used in our analysis, namely the matching to a general data set including all published stereotyped subsets instead of the auto-matching performed by those Authors. We demonstrated a significant association between IGHV status and stereotyped sequences and confirmed the finding that #1 is the most frequent subset identified so far. Finally, we were able to identify a series of 23 novel putative subsets that will require further confirmation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Microarray Gene Expression Profiling of B-Cell Chronic Lymphocytic Leukemia Subgroups Defined by Genomic Aberrations and VH Mutation Status

Journal of Clinical Oncology ◽

10.1200/jco.2004.12.133 ◽

2004 ◽

Vol 22 (19) ◽

pp. 3937-3949 ◽

Cited By ~ 142

Author(s):

Christian Haslinger ◽

Norbert Schweifer ◽

Stephan Stilgenbauer ◽

Hartmut Döhner ◽

Peter Lichter ◽

...

Keyword(s):

Gene Expression ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Differentially Expressed Genes ◽

Expression Profiling ◽

Lymphocytic Leukemia ◽

Differentially Expressed ◽

Mutational Status ◽

Genomic Aberrations ◽

Cell Chronic Lymphocytic Leukemia

Purpose Genomic aberrations and mutational status of the immunoglobulin variable heavy chain (VH) gene have been shown to be among the most important predictors for outcome in patients with B-cell chronic lymphocytic leukemia (B-CLL). In this study, we report on differential gene expression patterns that are characteristic for genetically defined B-CLL subtypes. Materials and Methods One hundred genetically well-characterized B-CLL samples, together with 11 healthy control samples, were analyzed using oligonucleotide arrays, which test for the expression of some 12,000 human genes. Results Aiming at microarray-based subclassification, class predictors were constructed using sets of differentially expressed genes, which yielded in zero or low misclassification rates. Furthermore, a significant number of the differentially expressed genes clustered in chromosomal regions affected by the respective genomic losses/gains. Deletions affecting chromosome bands 11q22-q23 and 17p13 led to a reduced expression of the corresponding genes, such as ATM and p53, while trisomy 12 resulted in the upregulation of genes mapping to chromosome arm 12q. Using an unsupervised analysis algorithm, expression profiling allowed partitioning into predominantly VH-mutated versus unmutated patient groups; however, association of the expression profile with the VH mutational status could only be detected in male patients. Conclusion The finding that the most significantly differentially expressed genes are located in the corresponding aberrant chromosomal regions indicates that a gene dosage effect may exert a pathogenic role in B-CLL. The significant difference in the partitioning of male and female B-CLL samples suggests that the genomic signature for the VH mutational status might be sex-related.

Download Full-text