Insulin Growth Factor 1 Receptor Expression Is Associated with NOTCH1 Mutation, Trisomy 12 and Aggressive Clinical Course in Chronic Lymphocytic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3314-3314
Author(s):  
Francesco Maura ◽  
Laura Mosca ◽  
Fabris Sonia ◽  
Giovanna Cutrona ◽  
Serena Matis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Strong Association ◽  
Lymphocytic Leukemia ◽  
Wild Type ◽  
Insulin Growth Factor ◽  
Cd38 Expression ◽  
Mutational Status ◽  
Hematological Cancers ◽  
Trisomy 12 ◽  
Igf1r Expression

Abstract Insulin growth factor 1 receptor (IGF1R) is emerging as an important gene in many solid and hematological cancers and its over expression has been reported to be associated with aggressive disease and pharmacologic resistance. Specifically, the IGF1R-IGF1-2 interaction was recently described to be involved in the constitutive activation of many important cell signaling such as NOTCH1 and PI3K/Akt pathways that play a key role in many solid and hematological cancers. In this study we performed a clinical and biological investigations about the role of IGF1R expression in a large and representative prospective series of chronic lymphocytic leukemia (CLL) in Binet A clinical stage enrolled in observation O-CLL1 protocol (clinicaltrial.gov identifier NCT00917540). Total RNA extraction, preparation of DNA single-stranded sense target, and hybridization to gene expression profiling arrays were carried out according to manufacturer’s protocols in 217 CLL patients enrolled in the multicentre O-CLL1 protocol. Gene expression data has been deposited in the National Centre for Biotechnology Information’s Gene Expression Omnibus database http://www.ncbi.nlm.mih.gov/geo and are accessible through series accession number GSE51529. High IGF1R expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high ZAP-70 and CD38 expression (p<0.0001) and unfavorable cytogenetic deletion [i.e. del(11)(q23) and del(17)(p13)], particularly with del(11)(q23) (p=0.03). Cases with del(13)(q14) as single lesion were characterized by the lowest IGF1R gene levels. On the contrary, among the most common cytogenetic aberrations, trisomy 12 showed stronger IGF1R expression compared with the other patients (p<0.0001) and this association was independent from IGHV mutational status. Patients with stereotyped HCDR3 sequences showed a greater IGF1R expression compared to not stereotyped HCDR3 (p=0.001) even if this can be related to the high frequency of IGHV-UM among stereotyped HCDR patients. Interestingly, subset #4 patients, who are known to exhibit an indolent clinical course and distinct biological profile, were also characterized by lower IGF1R expression compared to other M-IGHV and UM-IGHV patients. NOTCH1 c.7541_7542delCT mutation was investigated by next generation sequencing Roche 454 technology in 199 (92%) patients (Lionetti et al, BJH 2014). Globally, median depth of coverage was 1510x, ranging from 605 to 2842. Mutant allele frequency estimated by NGS ranged from 0.02% to 75% of total reads per sample. The presence of NOTCH1 mutation was confirmed by ASO-PCR and Sanger sequencing in all patients with allele burden higher > 0.7% (31; 15.5%) and 7% (19; 9.5%) respectively. We considered as mutated only the 31 patients in whom the presence of the dinucleotide deletion was confirmed by ARMS-PCR. Patients carrying NOTCH1 mutation were characterized by a greater IGF1R expression compared with wild type cases (p=0.002). In addition high IGF1R expression was not significantly different comparing patients with low and high NOTCH1 mutation burden. In order to avoid the bias represented by the strong association between NOTCH1 and trisomy 12, we compared the IGF1R expression between NOTCH1 mutated and wild type cases excluding trisomy 12, and confirmed the previous association (p=0.004). IGF1R expression represented a strong clinical prognostic factor in our CLL cohort: by Kaplan-Maier analysis we observed a significant time to first treatment stratification in all CLLs, in all IGHV-UM and in all IGHV-M patients (p<0.0001). Furthermore, IGF1R retained its significance in multivariate analysis with most important clinical and molecular prognostic factors (CD38 expression, unfavorable FISH and IGHV mutational status). Overall, our study shows the importance of IGF1R expression in CLL and its strong association with specific clinical and biological features, confirming the interest for the study of this gene as a potential prognostic factor and its possible role as a therapeutic target in a specific group of CLL patients carrying trisomy 12 and NOTCH1 mutations. Disclosures No relevant conflicts of interest to declare.

Co-Expression of CD38 with Zap-70 Is Predictive of Treatment Intervention in Patients (pts) with Early Stage Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4945-4945
Author(s):  
Jean-Gabriel Coignet ◽  
Swaminathan Padmanabhan ◽  
Rami Manochakian ◽  
Kena C. Miller ◽  
Paul Wallace ◽  
...  
Keyword(s):  
Early Stage ◽  
Lymphocytic Leukemia ◽  
Treatment Intervention ◽  
Symptomatic Disease ◽  
Cd38 Expression ◽  
Cytogenetic Aberrations ◽  
Mutational Status ◽  
Trisomy 12 ◽  
Aggressive Clinical Course ◽  
First Time

Abstract Introduction: CLL is a heterogeneous disease where advanced stage pts have compromised survival despite treatment, while early stage pts may not require any intervention. Based on current NCI-WG guidelines, most early stage (Rai stage 0 and 1) CLL pts do not require treatment until the development of progressive or symptomatic disease, though eventually over 70% of CLL pts will receive therapy. Currently, there are no markers to predict this subset of pts. Several markers of adverse prognosis, including ZAP-70+, CD38+, un-mutated IgVH gene and cytogenetic aberrations (17p-, 11q-, +12) have been identified. These markers are primarily studied in context of aggressive clinical course and survival outcome. The value of these markers to predict the necessity of treatment intervention in early stage CLL pts has not yet been completely evaluated. We investigated the expression pattern of Zap-70 and CD38 to examine their predictive value in identifying early stage CLL pts who will require therapeutic intervention. Methods & Results: 93 CLL pts were evaluated since 2002 at our institution. Flow cytometry was used to determine Zap-70 and CD38 expression on CD19+ CLL cells obtained from peripheral blood. Pts were considered to be positive for Zap-70 and CD38 expression if ≥ 20% and ≥ 30% of the cell stained for these proteins, respectively. For the Zap-70 analysis we used similar methodology as described by Crespo et al.1 Thirty-six (19M, 17F) pts had limited stage CLL, based on Rai staging criterion. Median age was 65 years (range 43–86) with stage 0 or 1 observed in 14 and 22 pts, respectively. Median time from diagnosis is 2 years (range &lt;1–17). Increased expression of either Zap-70 or CD38 was seen in 22 and 7 pts, respectively. Five (14%) pts were positive for both. All (100%) pts with concurrent increased expression of Zap-70 and CD38 required treatment (p=0.003), as compared to 30% of patients requiring treatment that were negative for both proteins or positive for only protein. Among the pts that required treatment 4 had 13q- and one had trisomy 12 on cytogenetic analysis. Conclusion: Our study, demonstrates for the first time, the clinical utility of CD38 and Zap-70 co-expression in determining the probability of treatment intervention in early stage CLL pts. Although the number of pts studied is small, our findings highlight a potentially important use of these markers in the management of early-stage CLL pts. These observations warrant validation in a larger cohort of pts early stage CLL pts as well as correlation with other prognostic markers such as cytogenetic and IgVH gene mutational status.

scholarly journals Subgroup-specific gene expression profiles and mixed epistasis in chronic lymphocytic leukemia

2021 ◽  
Author(s):  
Almut Luetge ◽  
Junyan Lu ◽  
Jennifer Huellin ◽  
Tatjana Walther ◽  
Leopold Sellner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Specific Gene ◽  
Molecular Heterogeneity ◽  
Mutational Status ◽  
Recurrent Mutations ◽  
Trisomy 12

Despite the extensive catalogue of recurrent mutations in chronic lymphocytic leukaemia (CLL), the diverse molecular driving events and the resulting range of disease phenotypes remain incompletely understood. To study the molecular heterogeneity of CLL, we performed RNA-sequencing on 184 CLL patient samples. Unsupervised analysis revealed two major independent axes of gene expression variation: the first one aligned with the mutational status of the immunoglobulin heavy variable (IGHV) genes, and concomitantly, with the three-group stratification of CLL by global DNA methylation pattern, and affected biological functions including B- and T-cell receptor signaling. The second one aligned with trisomy 12 status and affected chemokine signaling. Furthermore, we searched for differentially expressed genes associated with gene mutations and copy-number aberrations and detected strong signatures for TP53, BRAF and SF3B1, as well as for del(11)(q22.3), del(17)(p13) and del(13)(q14) beyond the dosage effect. We discovered strong non-additive effects (i.e., genetic interactions, or epistasis) of IGHV mutation status and trisomy 12 on multiple phenotypes, including the expression of 893 genes. Multiple types of epistasis were observed, including synergy, buffering, suppression and inversion. Our study reveals previously underappreciated gene expression signatures for (epi)genomic variants in CLL and the presence of epistasis between them. The findings will serve as a reference for a functional resolution of CLL molecular heterogeneity.

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

2001 ◽  
Vol 194 (11) ◽  
pp. 1639-1648 ◽  
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.

Analysis of B-CLL with Discordant ZAP-70 Expression and IgVH Mutational Status.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1194-1194 ◽  
Author(s):  
Remi Letestu ◽  
Magali Le Garff-Tavernier ◽  
Dominique Vaur ◽  
Michel Ticchioni ◽  
Fanny Baran-Marszak ◽  
...  
Keyword(s):  
B Cells ◽  
Somatic Mutations ◽  
Staining Procedure ◽  
Fish Analysis ◽  
Prognostic Information ◽  
Proliferation Markers ◽  
Cd38 Expression ◽  
Mutational Status ◽  
Trisomy 12 ◽  
Igvh Mutational Status

Abstract The clinical course of CLL is heterogeneous and presence or absence of IgVH somatic mutations has been correlated with stable and evolutive disease respectively. ZAP-70 protein expression was shown to be associated with unmutated IgVH genes in CLL and was proposed as a surrogate for mutational status. Recent studies of large number of cases have shown various percentage of discrepancy with respect to ZAP-70 expression and IgVH mutational status. As aggressive disease is not always associated with unmutated IgVH genes we aimed at better characterize the ZAP-70 discordant cases by determining the other prognostic factors such as cytogenetics, expression of CD38 and proliferation markers (thymidine kinase and sCD23). We investigated 292 patients with previously untreated B-CLL. Although several antiZAP-70 antibodies adapted to flow cytometry (FCM) are commercially available, staining procedure and interpretation of the results still remain controversial. Therefore, ZAP-70 expression was determined in the four participating centers by the same FCM method using the 2F3.2 mAb with indirect staining, and expression of the results was standardized. Moreover, ZAP-70 expression was investigated by RQ-PCR in 62 cases and by Western blot in 61 further cases on isolated B cells. The results obtained with these techniques were correlated with FCM data. ZAP-70 was found expressed in 141 cases, among which 38 cases (27%) exhibited ≥2% somatic mutations. Conversely, among the 151 ZAP-70 negative cases, only 6 cases (4%) were found unmutated (≥ 98% VH homology). We focused on the characteristics of the mutated ZAP-70 positive cases. Only 26/38 were in stage A at diagnosis. Incidence of CD38 expression >10% B cells was low (7/28 cases). Analysis of VH sequences pointed to the frequency of VH3-21 usage in 8/38 cases (21%) as compared to an expected 3% frequency in the French population. FISH analysis identified one case with del11q22.3, one case with del17p and two cases with trisomy 12. Del13q14 was present in half of cases. Proliferation markers were significantly higher in these cases than in ZAP-70 negative mutated cases, even among stage A patients. Follow-up of these patients is still too short for significant event free survival as compared with other groups of patients but the number of evolutive Binet stage A patients and advanced B and C stages was already higher than among the ZAP negative mutated cases. In conclusion, in our hands, ZAP-70 was almost always expressed in unmutated cases (103/109). Among mutated cases, ZAP-70 expression was present in 38/183 cases (21%), and brought prognostic information, independently of CD38 or chromosomal alterations. The correlation between ZAP-70 and proliferation markers suggests that ZAP-70 expression may result in a survival advantage of the malignant cells independently of the mutational status.

Angiopoietin-2 Expression in B-Cell Chronic Lymphocytic Leukemia: Association with Clinical Outcome and Immunoglobulin Heavy-Chain Mutational Status.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2780-2780
Author(s):  
Rossana Maffei ◽  
Silvia Martinelli ◽  
Ilaria Castelli ◽  
Rita Santachiara ◽  
Elena Morandi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Clinical Outcome ◽  
Heavy Chain ◽  
Lymphocytic Leukemia ◽  
Adverse Clinical Outcome ◽  
Mutational Status ◽  
Angiopoietin 2 ◽  
Normal Controls ◽  
Cell Chronic Lymphocytic Leukemia

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.

Relative Prognostic Significance of ZAP-70 and IGHV1-69 Expression in Chronic Lymphocytic Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3891-3891
Author(s):  
Laura Z. Rassenti ◽  
Emanuela M. Ghia ◽  
Lillian Werner ◽  
Donna Neuberg ◽  
George F. Widhopf ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Regression Model ◽  
Heavy Chain ◽  
Research Funding ◽  
Cox Regression ◽  
Strong Association ◽  
Lymphocytic Leukemia ◽  
Cox Regression Model ◽  
Clinical Behavior ◽  
Trisomy 12

Abstract Abstract 3891 The leukemia B cells of patients (pts) with chronic lymphocytic leukemia (CLL) express a restricted repertoire of immunoglobulin heavy chain variable region (IGHV) genes. Moreover, certain IGHV genes appear over-represented in this repertoire. Among these, IGHV1-69 was first identified as most frequently expressed, being used by the CLL cells of nearly 20% of all pts (PNAS, 86:5913–7, 1989). CLL cells most frequently express IGHV1-69 without somatic mutations and often with restricted D and JH segment use, providing for stereotypic motifs in the heavy chain third complementarity determining region (HCDR3). We addressed whether there were peculiar biologic or clinical features of pts with CLL that used IGHV1-69. For this, we studied 452 pts identified in a cohort of 2,866 followed by the CLL Research Consortium (CRC) found to have CLL cells that express IGHV1-69. This accounted for 16% of all pts. We found that 420 of 452 CLL samples (93%) express IGHV1-69 without somatic mutation (≥98% sequence homology with germline IGHV1-69), which is in significant contrast to the frequency use of UM IGHV among CLL samples that do not use IGHV1-69. As noted for CLL pts in general, there is a strong association between mutation status and clinical behavior. Among pts that use IGHV1-69, the 32 of 452 pts that use MU IGHV1-69 had a highly indolent clinical course, with a median time from diagnosis to initial treatment (TFS) of 17.3 years. This was significantly longer than the median TFS of 2.8 yrs for the 452 pts that used UM IGHV1-69 (p<0.0001). Among the pts that used UM IGHV1-69 we identified a stereotypic HDCR3 motif shared by more than one patient in 249/452 (55%) of the cases. Multivariate analysis failed to discriminate any significant differences in the median TFS of pts that used IGHV1-69 that had a stereotypic CDR3 motif versus pts who had an idiosyncratic HCDR3 (2.9 yrs vs 3.0 yrs, respectively p=0.14). Interphase FISH for common cytogenetic aberrations in CLL were available for 281 of the 452 cases. Among these, 58% of the cases had deletions at 17p (18%), 11q (23%), or trisomy 12 (17%). The remaining cases had no detectable chromosomal abnormalities (24%) or isolated deletion of 13q (18%). The CLL cells of all 452 pts were examined for ZAP-70. There was a strong association between the expression of ZAP-70 and use of UM IGHV1-69. However, the association between ZAP-70 expression and use of UM IGHV1-69 was not absolute. Only 70% with UM IGHV1-69 were ZAP-70 positive, as were 12.5% that used M IGHV1-69. Of all 452 pts that expressed IGHV1-69, 300 (66%) had ZAP-70 positive CLL cells. These pts had a median TFS of 2.3 yrs, which was significantly shorter than that of the remaining 152 pts with ZAP-70-negative CLL, who had a median TFS of 4.3 yrs (p<0.0001). Moreover, of the 420 pts that used UM IGHV1-69, 296 (70%) had CLL cells that expressed ZAP-70; these pts had a median TFS of 2.3 yrs. This was significantly shorter than the median TFS of pts with CLL cells that express UM IGHV1-69, but were ZAP-70 negative 4.1 yrs (p<0.0001). A Cox regression model revealed that although the presence of detectable chromosomal aberrations was associated to a shorter median TFS, ZAP-70 was a stronger predictor of short TFS (HR for 13q =1.1 p =0.03, HR for trisomy 12 =1.2 p =0.03, HR for 11q =1.6 p =0.03, HR for 17p =1.8, p =0.03) (HR for ZAP-70 positive = 1.8, p=0.0004). The Cox regression model was used to assess the associations of ZAP-70, and the use or not of the UM IGHV1-69 gene with TFS (p-value <0.05 were considered as significant). We investigated these associations using a previously published cohort of characterized 705 CLL pts (Blood.2008;112:1923). The HR associated with the expression of ZAP-70 (HR=3.2) (p<0.0001) was significantly higher than if either the UM IGHV1-69 or the cases with UM IGHV other than IGHV1-69 were incorporated into the model (HR=1.9 and HR=1.6 respectively, p=0.001). We conclude that cases that use IGHV1-69 are peculiar in that they more frequently use UM IGHV and appear to have a higher frequency of adverse cytogenetic features than CLL cases at large. In addition, we found that CLL-cell expression of ZAP-70 can segregate pts that use UM IGHV1-69 into subgroups with disparate clinical behavior, despite the fact that all patients use the same IGHV gene. Moreover, multivariable analyses revealed that ZAP-70 was strongest predictor of short TFS among all other considered prognostic parameters in this distinctive cohort of pts. Disclosures: Kipps: Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

Functional Somatic and Germline Variants in the NF-κB Pathway in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 560-560 ◽  
Author(s):  
Ma. Reina Improgo ◽  
Adam Kiezun ◽  
Yaoyu Wang ◽  
Lillian Werner ◽  
Petar Stojanov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Transcriptional Activity ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Lymphocytic Leukemia ◽  
Wild Type ◽  
Rt Pcr ◽  
Germline Variants

Abstract Abstract 560 Nuclear factor kappa B (NF-κB) encompasses a family of transcription factors involved in oncogenic processes including cellular proliferation and apoptotic inhibition. Constitutive activation of NF-κB has been observed in hematologic malignancies and is thought to confer resistance to chemotherapeutic agents. Here, we examine the role of the NF-κB pathway in chronic lymphocytic leukemia (CLL). Whole-exome sequencing was performed using tumor and matched germline DNA from 167 CLL patients. We identified 51 patients (30%) carrying 53 non-silent somatic variants in genes of the canonical NF-κB pathway, which consists of 272 genes as defined by the Ingenuity Pathway Analysis tool. Of the 99 patients whose germline sequences have been analyzed to date, 27 patients (27%) carry 34 non-silent germline variants in NF-κB pathway genes. A total of 67 patients (40%) have at least one non-silent somatic or germline variant. Variants in the NFKB1 gene, itself, were also observed: a somatic variant, H66R, found in two patients, and two germline variants, Y89F and R849W, each found in one patient. To evaluate the functional consequences of the NFKB1 variants, we performed site-directed mutagenesis to generate full-length NFKB1 cDNAs encoding these variants. We subsequently measured transcriptional activity of wild-type and mutant NFKB1 via luciferase assays in HEK293T cells using reporter cassettes containing the NFKB1 response element. Transcriptional activity of the three NFKB1 variants was found to be at least 2-fold higher than that of wild-type NFKB1 (p<0.0001). We further hypothesized that this increased transcriptional activity would lead to increased expression of NFKB1 downstream target genes. Analysis of gene expression profiles from Affymetrix HG-U133 Plus 2.0 Arrays of 65 CLL patient samples showed that the NFKB1 downstream targets CCL3, CCL4, and CD69 are upregulated in NFKB1 variants. To validate these results, we performed quantitative RT-PCR in patients with (n=3) or without (n=9) NFKB1 variants and confirmed upregulation of CCL3 (p=0.0286), CCL4 (p=0.0384), and CD69 (p=0.0263). Direct transfection of HEK293T cells with NFKB1 variants also resulted in a 3.3-fold upregulation of CCL3 (p=0.05). To test the hypothesis that deregulation of the NF-κB pathway is a key mechanism in CLL, we compared gene expression profiles of NF-κB pathway genes between CLL patient samples (n=146) and normal B cells (n=16) and found overall upregulation of the NF-κB pathway in CLL (Kolmogorov-Smirnov test, p=2.2e-16). K-means clustering and principal component analysis (PCA) further revealed that CLL patients can be divided into two subgroups exhibiting differential magnitude of NF-κB pathway upregulation. Studies in progress aim to identify the clinical significance of these subgroups. Finally, we assessed the effect of inhibiting the NF-κB pathway using the cell permeant NF-κB inhibitor, SN50. We performed Annexin V/PI staining 24 hours post-treatment in CLL cells with (n=9) or without (n=3) NF-κB pathway variants. SN50 increased cell death 1.8-fold in all cells tested (p<0.0001). Quantitative RT-PCR also showed a 59% decrease in expression of CCL3 one hour post-treatment, confirming inhibition of the NF-κB pathway. In conclusion, our findings demonstrate that a high proportion of CLL patients harbor somatic and germline variants in NF-κB pathway genes, some of which appear to be functional. Furthermore, the NF-κB pathway is upregulated in CLL and pharmacological inhibition of the pathway leads to increased cancer cell death. Functional characterization of NF-κB pathway variants offers mechanistic insight into the disease, providing novel targets for therapy. Disclosures: No relevant conflicts of interest to declare.

Vegf and bFGF Single-Nucleotide Polymorphisms In Chronic Lymphocytic Leukemia: Impact On Susceptibility

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5287-5287
Author(s):  
Sandra Ballester ◽  
Begoña Pineda ◽  
Eduardo Tormo ◽  
Blanca Navarro ◽  
Ariadna Perez ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Lymphocytic Leukemia ◽  
P Value ◽  
Nucleotide Polymorphisms ◽  
Exact Test ◽  
Cd38 Expression ◽  
Mutational Status ◽  
The Individual

Abstract Background B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with a highly variable clinical outcome. Recent studies have identified a number of different molecular prognostic markers (including mutational status of the IgVH gene, ZAP70 and CD38 expression) that allow to discriminate patients in prognostic subgroups. However, different expression patterns of angiogenic factors as VEGF, VEGFR1 and bFGF have been related with B-CLL susceptibility and treatment requirements. We have analyzed the polymorphisms: -710 C/T in VEGFR1, rs1109324, rs1547651, rs3025039 (936C/T) and rs833052 in VEGF and rs1449683 (223 C/T) in bFGF in order to determine the possible association with susceptibility in B-CLL. Methods Peripheral blood samples from 230 B-CLL patients and 476 healthy controls were genotyped using probes TaqMan SNP Genotyping Assays. Samples were providing from the Hospital Clinic of Valencia. Four SNPs in the VEGF gene, one SNP in the bFGF gene and one SNP in the VEGFR1 gene were evaluated. Statistical analysis was performed using SNPStats program (Catalan Institute of Oncology) and Fisher's exact test was applied to evaluate the significance. Results We have observed an increased frequency of the T allele in the rs1449683 SNP [OR 1.62 (95% CI: 0.98-2.66) p-value =0.063] and in the rs1547651 SNP [OR 0.72 (95% CI: 0.51-1.03), p-value=0.072] in our B-LLC patients when compared to control subjects. Moreover we observed that T allele carriers of rs3025039 (VEGF) have a significant protective effect concerning this disease [OR 0.59 (95% CI: 0.39-0.89) p-value=0.009]. Conclusion Our data indicate an increased frequency of the T allele in polymorphisms rs1449683 (bFGF) and rs1547651 (VEGF) in the group of patients, which possibly account for the individual susceptibility to develop B-CLL. On the other hand the data provided suggest that the T allele of VEGF rs3025039 is likely important genetic marker of susceptibility to B-CLL. Further studies regarding the role of pro-angiogenic markers in B-CLL would be beneficial to help elucidate pathogenic pathways in this disease. Disclosures: No relevant conflicts of interest to declare.

Revisiting Hypogammaglobulinemia in Chronic Lymphocytic Leukemia: A Combined Clinicobiological Approach

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5633-5633 ◽  
Author(s):  
Panagiotis Baliakas ◽  
Aliki Xochelli ◽  
Eva Minga ◽  
Anastasia Hadzidimitriou ◽  
Vassiliki Douka ◽  
...  
Keyword(s):  
Clinical Stage ◽  
Normal Serum ◽  
Lymphocytic Leukemia ◽  
Cd38 Expression ◽  
Serum Immunoglobulins ◽  
Cytogenetic Aberrations ◽  
Mutational Status ◽  
Ighv Gene ◽  
Immunoglobulin Subclasses ◽  
Notch1 Mutations

Abstract Chronic lymphocytic leukemia (CLL) is characterized by progressive hypogammaglobulinemia that can affect one or more immunoglobulin subclasses. Although many underlying mechanisms have been suggested, the pathogenesis of this phenomenon remains to be elucidated. In the present study, we revisit hypogammaglobulinemia in CLL through a combined clinicobiological approach aiming at identifying associations with particular disease profiles that would offer pathogenetic insight and guidance for further research. The study group included 412 CLL patients with available information about serum immunoglobulins either at diagnosis (n=380) or before treatment initiation (n=32). Patient characteristics were as follows: median age: 65 years; males/females: 266/146; Binet stage A: 272/335, unmutated IGHV genes (U-CLL): 140/412 cases (34%); CD38 expression: 59/330 cases (18%); clonotypic IG of the MD or G isotype: 250 and 43 cases, respectively; isolated del(13q): 64/136 (47%); trisomy 12: 18/183 (10%); del(11q): 18/186 (10%); del(17p): 11/189 (6%); NOTCH1 del7544-45/p.P2514Rfs*4: 8/219 (4%). With a median follow up of 5 years, 152/329 cases (46%) received treatment. Decreased immunoglobulin serum levels in at least one subclass were identified in 220/412 patients (53%), as follows: (i) decreased IgM, 172/412 cases (41%); (ii) decreased IgG, 78/412 cases (19%); (iii) decreased IgA, 100/412 cases (24%). In 36/412 cases (9%), a decrease in all serum immunoglobulin subclasses was noted. No statistically significant differences were identified between patients with normal serum immunoglobulin levels versus those with hypogammaglobulinemia regarding age, gender, disease burden at diagnosis, IGHV gene mutational status, CD38 expression, cytogenetic aberrations, NOTCH1 mutations and the incidence of a second malignancy. However patients with hypogammaglobulinemia exhibited increased need for treatment compared to patients with normal serum immunoglobulins (91/175 vs 61/154 respectively, p=0.025). Among cases with hypogammaglobulinemia, 90 (41%) and 26 (12%) exhibited isolated IgM and IgA subclass deficiency, respectively; isolated IgG decrease, was relatively rare (10/220 cases, 4%). Interestingly, when comparing isolated IgA versus other subclass deficiencies, statistically significant associations were identified with (i) advanced clinical stage (Binet B/C, Rai III/IV) (p=0.002); (ii) female gender (p=0.041); and, (iii) NOTCH1 mutations (p=0.004). A propos of the latter, it is noteworthy that in 5/8 (63%) mutant NOTCH1 cases with hypogammaglobulinemia, the affected subclass was IgA. Within our cohort, we identified cases belonging to one of three different, well characterized subsets with stereotyped B-cell receptor immunoglobulin (BcR IG), namely: (1) subset #1 (clan I IGHV genes/IGKV1(D)-39): U-CLL, clinically aggressive, n=12; (2) subset #2 (IGHV3-21/IGLV3-21), mixed IGHV mutational status, noted clinical aggressiveness, n=5; and, (3) subset #4, mutated IGHV4-34/IGKV2-30 BcR IG, clinically indolent, n=12. Notably, all subset #2 cases showed low levels of at least one serum subclass, while in 4/5 and 3/5 cases, two or all three immunoglobulin subclasses were affected. Although numbers are small, the incidence of hypogammaglobulinemia in subset #2 was significantly (p<0.05) higher compared to either subset #1 or subset #4). Univariate analysis revealed clinical stage, CD38 expression and IGHV mutational status as statistically important parameters (p<0.05) for both time-to-first–treatment (TTFT) and overall survival (OS); in contrast, hypogammaglobulinemia had no impact either on on TTFT or OS. In multivariate analysis, clinical stage and IGHV gene mutational status retained independent significance. In conclusion, abnormalities of serum immunoglobulins are detected in CLL patients with heterogeneous clinicobiological profiles, including different disease burden (clinical stage), cytogenetic aberrations and IGHV gene mutational status. However, certain observations reported herein, in particular the high incidence of hypogammaglobulinemia in subset #2 and the association of NOTCH1 mutations with IgA subclass deficiency, are noteworthy and indicate the need for research towards unraveling causal mechanisms among the observed interwined events. Disclosures No relevant conflicts of interest to declare.

Light Chain IgLV3-21 Is Associated with Poor Prognosis in Chronic Lymphocytic Leukemia Patients Independently of the Presence of Heavy Chain IgHV3-21

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3218-3218
Author(s):  
Basile Stamatopoulos ◽  
Thomas Smith ◽  
David Sims ◽  
Andreas Heger ◽  
Hélène Dreau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heavy Chain ◽  
Light Chain ◽  
Poor Prognosis ◽  
Mrna Translation ◽  
Lymphocytic Leukemia ◽  
The Other ◽  
Mutational Status ◽  
Initial Cohort ◽  
Independent Cohort

Abstract Background : Chronic Lymphocytic Leukemia (CLL) is characterized by a very heterogeneous clinical course and the immunoglobulin heavy-chain gene (IgHV) mutational status is currently considered the gold standard of prognostication: unmutated (UM) immunoglobulin heavy chain region (IgHV) is associated with a poor prognosis while patients with mutated IgHV (M) have more indolent disease. An exception are patients with IgHV3-21/IgLV3-21 who have poor prognosis irrespectively of the IgHV mutational status. Interestingly, IgHV3-21 is co-expressed with IgLV3-21 in the majority of cases. However, little is known about IgLV3-21: indeed this light chain has never been characterized independently of IgHV3-21 in terms of gene expression and prognostic impact. Methods: Based on a cohort of 32 patients with aggressive CLL, we used total RNAseq data of highly purified leukemic cells to define gene expression profiles and IgHV/IgLV rearrangements for each patient. Gene set enrichment analysis was correlated with treatment-free (TFS) and overall (OS) in the initial cohort of 32 patients and in an independent cohort of 255 patients where IgLV3-21 positivity was determined by real-time PCR and confirmed by Sanger sequencing. Results: Among the 32 initial CLL patients, 9 patients had an IgLV3-21 rearrangement, but only 1 patient carried the IgHV3-21 rearrangement. The other patients had VH1-24/69, VH3-9/66/23/48/53 and VH4-59 heavy chain rearrangements. RNAseq expression profiling yielded 1457 transcripts and 789 genes that were differentially expressed between IgLV3-21 patients and the other patients. Within the differentially expressed genes, 68% were upregulated while 32% were downregulated at least 1.5 fold. Gene set analysis revealed enrichment of genes related to translation enhancement (ribosome, translational reactome, peptide chain elongation, RNA metabolism - P<0.0001) and MYC target genes (P=0.0003), in line with recent finding showing that BCR stimulation can increase global mRNA translation including MYC-specific mRNA translation. In the initial cohort of 32 patients, IgLV3-21 patients had a median TFS of 17 months compared to 44 months in patients with another light chain (P=0.0270). Similarly, IgLV3-21 patients had a shorter median OS (88 months vs >192 months, P=0.0287).We validated these results in an independent cohort of 255 patients with 31 (12%) IgLV3-21 patients and 10 (4%) with IgHV3-21 (of which 8/10 also carried the light chain IgLV3-21 rearrangement). IgLV3-21 patients presented a median TFS/OS of 29/183 months compared to non IgLV3-21 patients who had a median TFS/OS of 88/292 months (P=0.0003/P=0.0142). In addition, when IgHV3-21 patients (n=10) were compared to IgLV3-21 only patients (n=23), no statistical difference was observed in terms of TFS or OS. Interestingly, if patients were classified according the IgHV mutational status, both IgHV3-21 and IgLV3-21 patients displayed a prognosis similar to UM patients: median TFS was 144, 32, 23, 48 months for M, UM, IgHV3-21 and IgLV3-21 patients, respectively (Figure A- P<0.0001). Similar results were observed for OS with a median OS of 292, 112, 128 and 241 months for M, UM, IgHV3-21 and IgLV3-21 patients, respectively (Figure B - P<0.0001). If all IgLV3-21 (n=31) were considered independently of their heavy chain, median TFS (29 months) were similar to UM patients (32 months, P=0.5536) and statistically different from M patients (144 months - P<0.0001, Figure C). Similar results were observed for OS (Figure D). Conclusions: Our results highlight for the first time the importance of light chain IgLV3-21 in CLL in terms of its differential gene expression profile and prognosis: IgLV3-21 is associated with a translational enhancement gene signature and confers a poor prognosis similar to UM patient irrespectively of the heavy chain IgHV3-21 or the IgHV mutational status. Figure Figure. Disclosures Schuh: Gilead: Consultancy, Honoraria, Research Funding; Roche, Janssen, Novartis, Celgene, Abbvie: Consultancy, Honoraria.

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