Exploring the Pathways to Chronic Lymphocytic Leukemia

Blood ◽  
2021 ◽  
Author(s):  
Freda Kathryn Stevenson ◽  
Francesco Forconi ◽  
Thomas J. Kipps
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tumor Cells ◽  
Cell Fate ◽  
Lymphocytic Leukemia ◽  
Designer Drugs ◽  
Clinical Behavior ◽  
Cellular Origin ◽  
Mutational Status ◽  
Survival Pathways ◽  
Accessory Cells

In chronic lymphocytic leukemia (CLL), increasing knowledge of the biology of the tumor cells has led to transformative improvements in our capacity to assess and treat patients. The dependence of tumor cells on surface immunoglobulin (Ig) receptor signaling, on survival pathways, and on accessory cells within the microenvironment has led to a successful double-barrelled attack with designer drugs. Studies have revealed that CLL should be classified based on the mutational status of the expressed IGHV sequences into two diseases, either unmutated (U) or mutated (M)-CLL, each with a distinctive cellular origin, biology, epigenetics/genetics, and clinical behavior. The origin of U-CLL lies among the natural antibody repertoire and dominance of IGHV1-69 reveals a superantigenic driver. In both U-CLL and M-CLL, a calibrated stimulation of tumor cells by self-antigens apparently generates a dynamic reiterative cycle as cells, protected from apoptosis, transit between blood and tissue sites. But there are differences in outcome, with the balance between proliferation and anergy favoring anergy in M-CLL. Responses are modulated by an array of microenvironmental interactions. Availability of T-cell help is a likely determinant of cell fate, the dependency on which varies between U-CLL and M-CLL, reflecting the different cells of origin, and affecting clinical behavior. Despite such advances, cell-escape strategies, Richter transformation, and immunosuppression remain as challenges, which only may be met by continued research into the biology of CLL.

Chronic Lymphocytic Leukemia Subset Expressing Mutated IGHV3-23 Has Peculiar Clinical and Biological Features.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1256-1256
Author(s):  
Riccardo Bomben ◽  
Michele Dal-Bo ◽  
Dania Benedetti ◽  
Daniela Capello ◽  
Francesco Forconi ◽  
...  
Keyword(s):  
Gene Ontology ◽  
Chronic Lymphocytic Leukemia ◽  
Tumor Suppressor ◽  
Heavy Chain ◽  
Tumor Suppressor Genes ◽  
Lymphocytic Leukemia ◽  
Clinical Behavior ◽  
Qrt Pcr ◽  
Biological Features ◽  
Mutational Status

Abstract Abstract 1256 Poster Board I-278 Introduction In the last years, the B cell receptor (BCR) has become a key molecule in chronic lymphocytic leukemia (CLL), given the correlation between mutational status of immunoglobulin heavy chain variable (IGHV) genes and disease prognosis. Recently, a fraction of CLL has been shown to preferentially express specific IGHV genes, often in a non-random combination with homologous heavy chain complementarity-determining region-3 (HCDR3) and peculiar light chains. Some of these stereotyped BCR mark CLL subsets with peculiar clinical behavior regardless of IGHV mutations. These data suggest a role for BCR in defining the clinical and biological features of CLL, also beyond the mutational status of IGHV genes. Patients and Methods A HCDR3-driven clustering of 1,426 IG sequences (1,398 patients) was performed using ClustalX(1.83). Time to treatment (TTT) intervals, Rai staging, IGHV mutational status, CD38, ZAP-70, and karyotype abnormalities evaluated by FISH were available for 617 patients. Gene expression profiling (GEP) and quantitative real-time PCR experiments (QRT-PCR) were performed on purified CLL cells. Results IGHV3-23 was totally absent in 71 identified stereotyped clusters despite being the second most frequently used IGHV gene, such distribution was significantly skewed (p<0.0001), compared with the distribution of IGHV genes belonging to stereotyped BCR clusters observed in our series. Although 109/134 IGHV3-23 were mutated (M), alignment of IGHV sequences revealed a high degree of conservation in the context of the 13 AA positions involved in superantigen binding by IGHV3 subgroup genes, suggesting that the majority of M IGHV3-23 cases maintained the capacity to mediate superantigen recognition and binding. Median TTT (73 months) of 43 M IGHV3-23 CLL was significantly shorter than median TTT (253 months, p=0.0153) of 333 M CLL, as well as of 326 M CLL in which 7 cases belonging to the bad prognosis IGHV3-21/IGLV3-21 cluster were excluded (253 months, p=0.0082). Multivariate Cox proportional hazard analyses selected IGHV3-23 usage (p=0.029), Rai stage (p<0.0001) and FISH group (p<0.0001) as independent markers of disease progression for 376 M CLL, and for the cohort in which 7 M CLL from the IGHV3-21/IGLV3-21 cluster were excluded. Comparing 5 M IGHV3-23 and 22 M non-IGHV3-23 CLL for their differential GEP, 212 genes were selected, 108 up-regulated and 104 down-regulated in M IGHV3-23 CLL. Using the “Gene-Ontology Tree Machine” platform, a set of growth/tumor suppressor genes (PDCD4, TIA1, RASSF5), all down-regulated in M IGHV3-23 CLL, was constantly found in several gene-ontology categories related to apoptosis. QRT-PCR confirmed a significant down-regulation of these genes in 15 M IGHV3-23 compared to 35 M non-IGHV3-23 CLL. Given the notion that PDCD4 and TIA1 are among the genes under control of miR-15a and miR-16-1 a “Gene Set Enrichment Analysis” carried out on the 212 differentially expressed genes, confirmed that M IGHV3-23 samples were significantly deprived in genes whose expression is under control of miR-15a and miR-16-1. Accordingly, QRT-PCR experiments performed on 15 M IGHV3-23 and 35 M non-IGHV3-23 CLL revealed significant higher levels of both miR-15a (p=0.0007) and miR-16-1 (p=0.0031) in M IGHV3-23 cases. No difference was found in the distribution of patients with 13q14 deletion between M IGHV3-23 CLL and M non-IGHV3-23 CLL (p=0.19). Considering the cases used for microRNA expression experiments (data available in 47/50 cases), 8/15 M IGHV3-23 CLL bore the 13q14 deletion in more than 20% of nuclei, against 19/32 cases in the group of M non-IGHV3-23 CLL (p=0.94). Conclusion Expression of IGHV3-23 marks a subset of M CLL with a worse prognosis; such a peculiar clinical behavior may be related to superantigen stimulation combined with down-regulation of specific growth/tumor suppressor genes and up-regulation of miR-15a and miR-16-1. Disclosures No relevant conflicts of interest to declare.

Impaired expression of p66Shc, a novel regulator of B-cell survival, in chronic lymphocytic leukemia

Blood ◽  
2010 ◽  
Vol 115 (18) ◽  
pp. 3726-3736 ◽  
Author(s):  
Nagaja Capitani ◽  
Orso Maria Lucherini ◽  
Elisa Sozzi ◽  
Micol Ferro ◽  
Nico Giommoni ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Apoptosis ◽  
Group Analysis ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Expression Of Genes ◽  
Mutational Status ◽  
Survival Pathways

Abstract Intrinsic apoptosis defects underlie to a large extent the extended survival of malignant B cells in chronic lymphocytic leukemia (CLL). Here, we show that the Shc family adapter p66Shc uncouples the B-cell receptor (BCR) from the Erk- and Akt-dependent survival pathways, thereby enhancing B-cell apoptosis. p66Shc expression was found to be profoundly impaired in CLL B cells compared with normal peripheral B cells. Moreover, significant differences in p66Shc expression were observed in patients with favorable or unfavorable prognosis, based on the mutational status of IGHV genes, with the lowest expression in the unfavorable prognosis group. Analysis of the expression of genes implicated in apoptosis defects of CLL showed an alteration in the balance of proapoptotic and antiapoptotic members of the Bcl-2 family in patients with CLL. Reconstitution experiments in CLL B cells, together with data obtained on B cells from p66Shc−/− mice, showed that p66Shc expression correlates with a bias in the Bcl-2 family toward proapoptotic members. The data identify p66Shc as a novel regulator of B-cell apoptosis which attenuates BCR-dependent survival signals and modulates Bcl-2 family expression. They moreover provide evidence that the p66Shc expression defect in CLL B cells may be causal to the imbalance toward the antiapoptotic Bcl-2 family members in these cells.

Identification of New Recurrent Lesions and Clinical Subsets by Genome-Wide DNA Profiling in Chronic Lymphocytic Leukemia with 17p Deletion.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4696-4696
Author(s):  
Francesco Forconi ◽  
Andrea Rinaldi ◽  
Ivo Kwee ◽  
Elisa Sozzi ◽  
Davide Rossi ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Prognostic Significance ◽  
Uniparental Disomy ◽  
Dna Profiling ◽  
Lymphocytic Leukemia ◽  
Extra Copy ◽  
Fish Genome ◽  
Clinical Behavior ◽  
Mutational Status ◽  
Genome Wide

Abstract Chronic lymphocytic leukemia (CLL) with deletion of 17p(p53) locus (17p- CLL) identifies the most aggressive CLL subset, due to active disease course and resistance to current treatments. Intrinsic mechanisms of the poor prognosis of 17p- CLL are scarcely known. In order to identify additional lesions occurring in 17p- CLL which contribute to prognosis, we performed genome-wide DNA profiling in 18 cases of 17p- CLL (median follow-up 25 months, range 4–107), using high-resolution single-nucleotide polymorphism (SNP) arrays (Affymetrix Human Mapping Nsp 250 k arrays). Chromosomal 11q, 13q or 17p deletions and trisomy 12 were determined by FISH. Tumor IGHV usage and mutational status were determined by PCR/sequencing of the tumor gDNA/cDNA. Median interval from diagnosis to progression (11 months, p=0.000122) and treatment (16 months, p=0.000827) were representative of the bad behavior of our 17p- CLL cases if compared to 95 non17p- CLL patients from our internal cohort (68 and 69 months, respectively). Interestingly, 3/18 (16%) 17p- CLL expressed the IGHV3–74 segment which is rare in normal B-cells (1,4%, p=0.02) and in non17p- CLL (2.1%, p=0.02). Tumor IGHV genes were unmutated in 10/18 and mutated in 8/18 17p- CLL. All cases carried 17p13.1 deletion in more than 60% nuclei, as assessed by FISH. Genome-wide DNA profiling was 99% concordant with FISH (71/72 data-points; one 13q deletion detected by FISH in 20% of the nuclei was missed by the microarray) and confirmed 17p deletion in all cases. Deletions at the 13q14.3 locus recurred in 50% cases. Only one case had an extra-copy of chromosome 12. No cases carried ATM deletion. Other recurrent lesions identified by genome-wide DNA profiling were: gains of 2p16.1-pter (30%), 3q24-qter (20%), 8q23.3-qter (30%), 17q21.2-q22 (30%), 14q32 (30%); losses of 8p12-pter (30%), 9q21.33-q22.1 (20%), 14q32 (40%), 20p12.1-pter (20%). Approximately half of the cases had concomitant losses of 8p or 17p and gains of the 8q or 17q regions, respectively, suggesting the presence of isochromosomes 8 or 17. Indeed, isochromosome 17 was evident in one case analyzed by conventional cytogenetics. Six patients had one or more regions of loss of heterozygosity (LOH) longer than 5 Mb in the absence of copy number reduction, suggestive of uniparental disomy. Several lesions, such as those involving chromosomes 3, 8 and 9, associate with poor clinical behavior in other B-cell tumors. Thus, we investigated the prognostic significance of the lesions identified in our series. Despite the small number of cases, we found that 17p- CLLs with loss of 8p12-pter, which contains tumor necrosis factor and defensin family genes, associated with worse treatment free interval from diagnosis (12 vs 46 months in 8p- vs non 8p- CLL, p=0.04). Overall, our analysis identified new recurrent genomic abnormalities at high frequencies that may specifically contribute to a different biologic and clinical behavior within 17p- CLL. Among these, isochromosomes 8 and 17 were particularly common, and 8p loss might identify a new aggressive subentity within 17p- CLL.

Phenotypic and Functional Features of Vγ9/Vδ2 T Cells in Chronic Lymphocytic Leukemia Are Strongly Correlated with the Mutational Status of the Immunoglobulin Heavy Chain Variable Region (IgVH) and Metabolic Activity of the Mevalonate Pathway in Tumor Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1131-1131
Author(s):  
Marta Coscia ◽  
Silvia Peola ◽  
Giorgia Matta ◽  
Francesca Pantaleoni ◽  
Myriam Foglietta ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Tumor Cells ◽  
Mevalonate Pathway ◽  
Γδ T Cells ◽  
Variable Region ◽  
Lymphocytic Leukemia ◽  
Effector Memory ◽  
Heavy Chain Variable Region ◽  
Mutational Status

Abstract Vγ9/Vδ2 (γδ) T cells have an important effector and regulatory role in innate and adaptive immunity against microbes, stressed cells and tumor cells. They represent less than 5% of peripheral blood lymphocytes, but can be activated and expanded in vitro by aminobisphosphonates (ABP) like zoledronic acid (Zol), as surrogates of their natural ligands. In this study, we have investigated the proliferative response of γδ T cells to Zol in 59 patients with chronic lymphocytic leukemia (CLL) at diagnosis. Proliferation of γδ T cells was observed in 30 patients (51%)(responders, R), whereas 29 patients (49%) were non-responders (NR). γδ T-cell subset distribution [e.g., naive (TN), central memory (TCM), effector memory (TEM), and terminally differentiated effector memory (TEMRA)] was well balanced in R patients, whereas TEM and TEMRA were largely predominant in NR patients. TEMRA of NR patients had a unique profile of NK receptors expression, characterized by the expression of the ILT4 inhibitory receptor, whereas R patients tended to have an higher expression of the costimulatory molecule NKG2D. Notably, HLA-G, a negative prognosticator in CLL and a well known ILT4 ligand, was more expressed in tumor cells of NR patients. This correlation prompted us to determine whether other tumor cell features were different in R and NR patients. Indeed, the latter showed significantly higher tumor cell counts, higher frequency of poor-risk cytogenetic abnormalities, and a significantly shorter median time to first treatment. Lastly, a very significant association was observed with the mutational status of the tumor immunoglobulin heavy chain variable region (IgVH), which was mutated (M) in 96% of R patients, and unmutated (UM) in 74% of NR patients. To gain further insight into this association, we evaluated the activity of the mevalonate pathway in tumor cells of M and UM patients. This pathway, that can be targeted by Zol, generates the phosphoantigens naturally recognized by γδ T cells. Bioinformatic and biochemical approaches showed that this pathway is more active in tumor cells of UM than M patients. Based on these data, we propose that tumor cells of UM CLL patients can more easily engage γδ T cells and, in the long run, exhaust their immune surveillance potential by driving their differentiation into functionally impaired TEMRA. In conclusion, we have identified a novel mechanism of immune escape which can contribute to the disease progression in UM CLL patients.

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.

The prognostic impact of autologous stem cell transplantation in patients with chronic lymphocytic leukemia: a risk-matched analysis based on the VH gene mutational status

Blood ◽  
2004 ◽  
Vol 103 (7) ◽  
pp. 2850-2858 ◽  
Author(s):  
Peter Dreger ◽  
Stephan Stilgenbauer ◽  
Axel Benner ◽  
Matthias Ritgen ◽  
Alexander Kröber ◽  
...  
Keyword(s):  
Stem Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Autologous Stem Cell Transplantation ◽  
Lymphocytic Leukemia ◽  
Study Entry ◽  
Prognostic Impact ◽  
Cox Regression Analysis ◽  
Mutational Status

Abstract To assess the therapeutic value of sequential high-dose therapy (SHDT) including autologous stem cell transplantation in chronic lymphocytic leukemia (CLL) we performed a risk-matched comparison between 66 patients who had undergone a uniform SHDT regimen and a database of 291 patients treated conventionally. Matching variables were age, Binet stage, IgVH (variable region of the immunoglobulin heavy chain) gene mutational status, and lymphocyte count. Forty-four pairs fully matched for all 4 variables were identified. Patient groups were well balanced for additional risk factors including adverse genomic abnormalities and CD38 expression. With an overall median follow-up time of 70 and 86 months, respectively, survival was significantly longer for the SHDT patients than for the conventionally treated patients when calculated from diagnosis (hazard ratio [HR] 0.39; P = .03 [log rank]) or from study entry (HR 0.32; P = .006). The benefit for the SHDT group remained significant when the analyses were restricted to those 58 patients who had an unmutated VH status. Cox regression analysis confirmed SHDT as independent favorable prognostic factor for survival from diagnosis (HR 0.38, P = .04) as well as from study entry (HR 0.38, P = .03). These data suggest a survival benefit for patients with poor-risk CLL receiving SHDT during the course of their disease. (Blood. 2004;103:2850-2858)

scholarly journals Chronic Lymphocytic Leukemia in Kuwait

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5467-5467
Author(s):  
Salem Alshemmari ◽  
Ramesh Pandita ◽  
Abdulaziz Hamadah ◽  
Ahmad Alhuraiji
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Board Of Directors ◽  
Mutational Analysis ◽  
Staging System ◽  
Lymphocytic Leukemia ◽  
Advisory Committees ◽  
Western Countries ◽  
Mutational Status ◽  
Study Results ◽  
History Of

Background :Chronic lymphocytic leukemia (CLL) is common malignancy in Western countries. However, little known about this disease entity in our area. This study exploring the biology in out patients' population. Method:Patients with confirmed CLL under IGHV and TP53 mutational analysis at presentation or during follow up. We also integrated other clinical and biological parameter in this study. Results: A total of 137 cases were analyzed, median age 61 years (range:34-89); 30% of the cases age was<55 years at presentation. There was 108 males vs. 29 females M:F ratio 3.7. Two patients gave a family history of CLL, while 1 patient gave a history of other lymphoproliferative disorders. Binet staging system available in 134 cases, A: 109 (81.3%), B: 12 (9%), C:13 (9.7%). B2 macroglobulin elevated in 40/112 (36%) cases and 10/103 (10%) had M-spike. CD38 positivity reported in 37/112 (33%) of cases. Cytogenetics data evaluable in 85 cases: isolated del(13q): 35%, isolated trisomy 12 (16.5%), del(11q) (4.5%), del(17p)(2.4%). IGHV mutational status mutated vs unmutated: 40% vs 60%. Cases with available treatment information on 132 cases. Fifty cases required treatment due to disease progression. First line treatment Bendamustine-Rituximab (BR) 3 cases, Fludarabine Cyclophosphamide Rituximab (FCR) 30 cases and Chlorambucil with anti-CD 20 antibody 6 cases. At the time of review, 3 cases on ibrutinib (2 in 3rdline and 1 case in the 4thline). Conclusion: This is the first study to shed light on CLL in our area. There are biological differences between our patients' population and the western countries. Disclosures Pandita: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Total Proteome Analysis Identifies Migration Defects As a Major Pathogenetic Factor in IGHV-Unmutated Chronic Lymphocytic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 718-718 ◽  
Author(s):  
Gina L Eagle ◽  
Rosalind E Jenkins ◽  
Kathleen J Till ◽  
Jithesh Puthen ◽  
Ke Lin ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Computational Analysis ◽  
Lymphocytic Leukemia ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Data Set ◽  
Clinical Behaviour ◽  
Mutational Status ◽  
Significant Difference ◽  
Total Proteome

Abstract The mutational status of the immunoglobulin heavy chain variable region (IGHV) defines two clinically distinct forms of chronic lymphocytic leukemia (CLL) known as mutated (M-CLL) and un-mutated (UM-CLL). Patients with M-CLL usually have a favourable outcome whereas those with UM-CLL develop progressive disease and have shorter survival. However, the molecular mechanisms responsible for the more aggressive clinical behaviour associated with UM-CLL are not well understood. Here we describe the application of isobaric tags for relative and absolute quantification (iTRAQ) based mass spectrometry (MS) to analyse the total proteome of M-CLL and UM-CLL samples. This has enabled us to generate the largest quantity of proteomic information for CLL to date and, in particular, to directly compare the functions of differentially expressed proteins between UM-CLL and M-CLL cells through a systems biology approach. We isolated CLL cells from the peripheral blood from 18 CLL patients (9 UM-CLL, 9 M-CLL) and prepared cellular protein extracts which were digested and subjected to labelling with iTRAQ reagents, as previously described (Kitteringham et al, J Proteomics, 2010;73(8):1612-1631). Principal component analysis was used to assess variance across the data set generated by iTRAQ-MS. Statistical significance of the difference in the levels of expression of proteins between UM-CLL and M-CLL samples was determined using student T-test (2-tailed). Several differentially expressed proteins identified by iTRAQ-MS were also validated by immunoblotting. Computational analysis was performed to examine the functions of the differentially expressed proteins and their associated signalling pathways using the GeneGo pathway maps in the Metacore™ database (Thomson Reuters, NY, USA). Unsupervised clustering, based on the expression of 3521 identified proteins, separated CLL samples into two groups corresponding to IGHV mutational status. We identified 274 proteins that were differentially expressed between UM-CLL and M-CLL subgroups (p<0.05, Figure 1A). Hierarchical clustering based on the relative expression of differentially expressed proteins also separated individual CLL cases into two distinct clusters according to their IGHV status (Figure 1B). Computational analysis showed that 43 cell migration/adhesion pathways were significantly enriched (p<0.05) by 39 differentially expressed proteins, 35 of which were expressed at significantly lower levels in UM-CLL samples. Furthermore, UM-CLL cells under-expressed proteins associated with cytoskeletal remodelling and over-expressed proteins associated with transcriptional and translational activity. Taken together, these findings indicated that UM-CLL cells are less migratory and more adhesive than M-CLL cells, resulting in their retention in lymph nodes where they are exposed to proliferative stimuli. In agreement with this hypothesis, analysis of an extended cohort of 120 CLL patients revealed that twice as many patients with UM-CLL than M-CLL had documented lymphadenopathy (50% v 24%; P<0.01). The association between UM-CLL and lymphadenopathy was not simply a reflection of increased tumour burden as there was no significant difference in the leukocyte count between the two groups (medians of 37 x 109/L and 28 x 109/L, respectively; P>0.05). In addition, other pathways that promote cell survival and proliferation in UM-CLL cells were also enriched by the differentially expressed proteins. These include the immune response pathway involving B-cell receptor (BCR) signalling (P=0.006), the endoplasmic reticulum (ER) stress response pathway (P=0.035) and the Wnt signalling pathway (P=0.006). Our study has shown that quantitative analysis of the total proteome by iTRAQ-MS was able to separate individual CLL cases according to IGHV status and explained the more aggressive clinical behaviour of UM-CLL and its particular sensitivity to novel therapeutic agents that induce anatomical displacement from the lymph node microenvironment, such as ibrutinib and idelalisib. Moreover, in keeping with the ability of proteomics to detect alterations in gene expression resulting from both transcriptional and post-transcriptional mechanisms, the study illustrates the considerable potential of iTRAQ-MS coupled with computational analysis to elucidate pathogenetic mechanisms and indicate therapeutic strategies in cancer. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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