Increased frequency of CD4+ PD-1+ HLA-DR+ T cells is associated with disease progression in CLL.

Chronic lymphocytic leukaemia (CLL) patients often have abnormal expansions of CD4+ and CD8+ T cells and this can be associated with progressive disease. To characterise the key T-cell populations involved in this phenomenon, we used flow cytometry and 11 phenotypic markers to study 74 CLL patients and 14 controls. T cells of CLL patients were more phenotypically complex than those of healthy controls with significant increases in the frequencies of CD4 and CD8 memory T cells expressing exhaustion-, activation- and senescence-associated markers. Multivariate analysis of 111 different T-cell subsets showed that high frequencies of four subsets (three CD8 and one CD4) were associated with shorter progression-free survival. The most significant association was with CD4+ HLA-DR+ PD-1+ T cells, and patients could be stratified into high- and low-risk groups based on the frequency of these T cells. The expansion of this CD4+ subset could not be accounted for by age, cytomegalovirus infection or increases in Treg cells. Overall, these results highlight two relatively simple biomarkers, percentage CD8+ and percentage CD4+ PD-1+ HLA-DR+ T cells, which can be used to risk-stratify CLL patients, independent of other tumour-associated markers. They also provide further evidence for the pivotal role of T cells in modulating the pathology of CLL.

Genetic correlation between multiple myeloma and chronic lymphocytic leukaemia provides evidence for shared aetiology.

The clustering of different types of B-cell malignancies in families raises the possibility of shared aetiology. To examine this, we performed cross-trait linkage disequilibrium (LD)-score regression of multiple myeloma (MM) and chronic lymphocytic leukaemia (CLL) genome-wide association study (GWAS) data sets, totalling 11,734 cases and 29,468 controls. A significant genetic correlation between these two B-cell malignancies was shown (Rg = 0.4, P = 0.0046). Furthermore, four of the 45 known CLL risk loci were shown to associate with MM risk and five of the 23 known MM risk loci associate with CLL risk. By integrating eQTL, Hi-C and ChIP-seq data, we show that these pleiotropic risk loci are enriched for B-cell regulatory elements and implicate B-cell developmental genes. These data identify shared biological pathways influencing the development of CLL and, MM and further our understanding of the aetiological basis of these B-cell malignancies.

Telomere length is an independent prognostic marker in MDS but not in de novo AML.

Telomere dysfunction is implicated in the generation of large-scale genomic rearrangements that drive progression to malignancy. In this study we used high-resolution single telomere length analysis (STELA) to examine the potential role of telomere dysfunction in 80 myelodysplastic syndrome (MDS) and 95 de novo acute myeloid leukaemia (AML) patients. Despite the MDS cohort being older, they had significantly longer telomeres than the AML cohort (P < 0·0001) where telomere length was also significantly shorter in younger AML patients (age <60 years) (P = 0·02) and in FLT3 internal tandem duplication-mutated AML patients (P = 0·03). Using a previously determined telomere length threshold for telomere dysfunction (3·81 kb) did not provide prognostic resolution in AML [Hazard ratio (HR) = 0·68, P = 0·2]. In contrast, the same length threshold was highly prognostic for overall survival in the MDS cohort (HR = 5·0, P < 0·0001). Furthermore, this telomere length threshold was an independent parameter in multivariate analysis when adjusted for age, gender, cytogenetic risk group, number of cytopenias and International Prognostic Scoring System (IPSS) score (HR = 2·27, P < 0·0001). Therefore, telomere length should be assessed in a larger prospective study to confirm its prognostic role in MDS with a view to integrating this variable into a revised IPSS.

Telomere length is a critical determinant for survival in multiple myeloma.

The variable clinical outcomes of Multiple Myeloma (MM) patients are incompletely defined by current prognostication tools. We examined the clinical utility of high-resolution telomere length analysis as a prognostic marker in MM. Cohort stratification, using a previously determined length threshold for telomere dysfunction, revealed that patients with short telomeres had a significantly shorter overall survival (P < 0·0001; HR = 3·4). Multivariate modelling using forward selection identified International Staging System (ISS) stage as the most important prognostic factor, followed by age and telomere length. Importantly, each ISS prognostic subset could be further risk-stratified according to telomere length, supporting the inclusion of this parameter as a refinement of the ISS.

Tumor cell migration is inhibited by a novel therapeutic strategy antagonizing the alpha-7 receptor.

A 14mer peptide (T14) derived from the C-terminus of acetylcholinesterase (AChE) selectively activates metastatic breast cancer cells via the alpha-7 nicotinic receptor (α7 nAChR). This naturally occurring peptide is also present in brain, is elevated in Alzheimer's disease, and is antagonised by a cyclized variant (NBP-14). Here we investigated the effects of NBP-14 in six different cancer cell lines, primary leukemia B-cells and normal B-cells. All cells tested expressed α7 nAChR, intracellular and extracellular T14. However, NBP-14 showed low toxicity and weak anti-proliferative effects in the majority of the cell lines and was even less toxic in normal B-cells when compared to primary chronic lymphocytic leukemia cells (P < 0.001). Given the potential role of T14 peptide in metastasis, we next investigated the effects of NBP-14 on tumor cell migration, where it caused a dose-dependent reduction. The extent of NBP-14 inhibition positively correlated with the migration of the cells (r2 = 0.45; P = 0.06). Furthermore, NBP-14 preferentially inhibited the migration of primary leukemia cells when compared with normal B-cells (P = 0.0002); when the normal B-cell data was excluded, this correlation was strengthened (r2 = 0.80; P = 0.006). Importantly, the constitutive α7 nAChR expression positively correlated with intracellular T14 levels (r2 = 0.91; P = 0.0003) and inversely correlated with extracellular T14 levels in the cell culture supernatants (r2 = -0.79; P = 0.034). However, in the presence of NBP-14, α7 nAChR expression was reduced (P = 0.04) and the most migratory cells showed the largest reduction in expression. In conclusion, NBP-14-mediated antagonism of the α7 nAChR offers a novel therapeutic strategy with the potential to inhibit tumor cell migration.

Genome-wide association analysis of chronic lymphocytic leukaemia, Hodgkin lymphoma and multiple myeloma identifies pleiotropic risk loci.

B-cell malignancies (BCM) originate from the same cell of origin, but at different maturation stages and have distinct clinical phenotypes. Although genetic risk variants for individual BCMs have been identified, an agnostic, genome-wide search for shared genetic susceptibility has not been performed. We explored genome-wide association studies of chronic lymphocytic leukaemia (CLL, N = 1,842), Hodgkin lymphoma (HL, N = 1,465) and multiple myeloma (MM, N = 3,790). We identified a novel pleiotropic risk locus at 3q22.2 (NCK1, rs11715604, P = 1.60 × 10-9) with opposing effects between CLL (P = 1.97 × 10-8) and HL (P = 3.31 × 10-3). Eight established non-HLA risk loci showed pleiotropic associations. Within the HLA region, Ser37 + Phe37 in HLA-DRB1 (P = 1.84 × 10-12) was associated with increased CLL and HL risk (P = 4.68 × 10-12), and reduced MM risk (P = 1.12 × 10-2), and Gly70 in HLA-DQB1 (P = 3.15 × 10-10) showed opposing effects between CLL (P = 3.52 × 10-3) and HL (P = 3.41 × 10-9). By integrating eQTL, Hi-C and ChIP-seq data, we show that the pleiotropic risk loci are enriched for B-cell regulatory elements, as well as an over-representation of binding of key B-cell transcription factors. These data identify shared biological pathways influencing the development of CLL, HL and MM. The identification of these risk loci furthers our understanding of the aetiological basis of BCMs.

Phenotype and immune function of lymph node and peripheral blood CLL cells are linked to transendothelial migration.

Several lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here, we demonstrate that lymph node (LN)-derived CLL cells possess a distinct phenotype, and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared with paired peripheral blood (PB) samples. LN-derived CLL cells manifest a proliferative, CXCR4(dim)CD5(bright) phenotype compared with those in the PB and higher expression of T-cell activation molecules including CD80, CD86, and HLA-D-related (DR). In addition, LN-CLL cells have higher expression of α4ß1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4(dim)CD5(bright) with higher CD49d, CD80, CD86, and HLA-DR compared with those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49d(hi) CLL cells showed an enhanced capacity to activate T cells compared with CD49d(lo) subpopulations from the same patient. Thus, although PB-CLL cells have a reduced capacity to form immune synapses and activate CD4(+) T cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunologic function is not only modulated by microenvironmental interactions but is also a feature of a subpopulation of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T cells.

A CD21 low phenotype, with no evidence of autoantibodies to complement proteins, is consistent with a poor prognosis in CLL.

B-cell chronic lymphocytic leukemia (CLL) is characterized by differential BCR signaling and autoimmune complications. Complement modulates B-cell function via C3d and CD21 cross-linked to the B-cell receptor (BCR). We hypothesized that CD21 contributes to BCR signaling and participates in the autoimmunity associated with CLL. We analyzed CD21 expression on 106 CLL patient samples and matched serum from 50 patients for the presence of soluble CD21 and autoantibodies to CR2, CR1, MCP and FH. CD21 expression on CLL B-cells was significantly lower than that expressed on B-cells from age-matched controls (P < 0.0001) and was inversely correlated with soluble CD21 (r2 = -0.41). We found no evidence of autoantibody to any complement regulator. Low CD21 expression correlated to prognostic subsets of CLL patients, i.e. cases with unmutated IGHV genes (P = 0.0006), high CD38 (P = 0.02) and high ZAP70 expression (P = 0.0017). Low CD21 expression was inversely correlated to the levels of phosphotyrosine induced in CLL cells following BCR ligation with αIgM (r2 = -0.21). Importantly, lower CD21 expression was also predictive for reduced overall survival (P = 0.005; HR = 2.7). In conclusion, we showed that reduced expression of CD21 on CLL B-cells appears functionally relevant and was associated with poor clinical outcomes.

Telomere analysis to predict chronic lymphocytic leukemia outcome: a STELA test to change clinical practice?

Defining the prognosis of individual chronic lymphocytic leukemia patients remains a significant clinical challenge. Consequently, there is a need to identify tests that can provide reliable personalized risk assessments. Here we discuss the problems associated with the currently used prognostic markers and emphasize the potential for using high-resolution telomere length analysis (STELA) for the accurate prediction of clinical outcome. Given the development of targeted, less toxic therapeutics in chronic lymphocytic leukemia, it is crucial to accurately identify those patients who might benefit from early treatment and equally those who may not require treatment at all. In this context, there is also a clear need for dependable predictive markers of response to drugs so that optimal treatment decisions can be made for individual patients.

CD8+ T-cell recognition of a synthetic epitope formed by t-butyl modification.

We set out to clone Bax-specific CD8+ T-cells from peripheral blood samples of primary chronic lymphocytic leukemia patients. A number of clones were generated using a Bax peptide pool and their T-cell epitope was mapped to two peptides sharing a common 9-aa sequence (LLSYFGTPT), restricted by HLA-A*0201. However, when these T-cell clones were tested against highly purified syntheses (>95%) of the same peptide sequence, there was no functional response. Subsequent mass spectrometric analysis and HPLC fractionation suggested that the active component in the original crude peptide preparations (77% pure) was a peptide with a tert-butyl (tBu) modification of the tyrosine residue. This was confirmed by modification of the inactive wild type (wt) sequence to generate functionally active peptides. Computer modeling of peptide:HLA-A*0201 structures predicted that the tBu modification would not affect interactions between peptide residues and the HLA binding site. However these models did predict that the tBu modification of tyrosine would result in an extension of the side chain out of the peptide-binding groove up towards the TCR. This modified product formed <1% of the original P603 crude peptide preparation and <0.05% of the original 23 peptide mixture used for T-cell stimulation. The data presented here, illustrates the potential for chemical modifications to change the immunogenicity of synthetic peptides, and highlights the exquisite capacity of TCR to discriminate between structurally similar peptide sequences. Furthermore this study highlights potential pitfalls associated with the use of synthetic peptides for the monitoring and modulating of human immune responses. This article is protected by copyright. All rights reserved.

Telomere dysfunction accurately predicts clinical outcome in chronic lymphocytic leukaemia, even in patients with early stage disease.

Defining the prognosis of individual cancer sufferers remains a significant clinical challenge. Here we assessed the ability of high-resolution single telomere length analysis (STELA), combined with an experimentally derived definition of telomere dysfunction, to predict the clinical outcome of patients with chronic lymphocytic leukaemia (CLL). We defined the upper telomere length threshold at which telomere fusions occur and then used the mean of the telomere 'fusogenic' range as a prognostic tool. Patients with telomeres within the fusogenic range had a significantly shorter overall survival (P < 0·0001; Hazard ratio [HR] = 13·2, 95% confidence interval [CI] = 11·6-106·4) and this was preserved in early-stage disease patients (P < 0·0001, HR=19·3, 95% CI = 17·8-802·5). Indeed, our assay allowed the accurate stratification of Binet stage A patients into those with indolent disease (91% survival at 10 years) and those with poor prognosis (13% survival at 10 years). Furthermore, patients with telomeres above the fusogenic mean showed superior prognosis regardless of their IGHV mutation status or cytogenetic risk group. In keeping with this finding, telomere dysfunction was the dominant variable in multivariate analysis. Taken together, this study provides compelling evidence for the use of high-resolution telomere length analysis coupled with a definition of telomere dysfunction in the prognostic assessment of CLL.

Development and characterization of a physiologically relevant model of lymphocyte migration in chronic lymphocytic leukemia.

There is growing evidence that lymphocyte trafficking contributes to the clinical course of chronic lymphocytic leukemia (CLL), but to date, only static in vitro cultures have been used to study these phenomena. To address this lack of data, we have developed a dynamic in vitro model in which CLL cells experience shear forces equivalent to those in capillary beds and are made to flow through capillary-like hollow fibers lined with endothelial cells. CLL cells treated in this way increased their expression of CD62L and CXCR4 (both P < .0001) and of CD49d and CD5 (both P = .003) directly as a result of the shear force. Furthermore, CLL cells migrated through the endothelium into the "extravascular" space (mean migration, 1.37% ± 2.14%; n = 21). Migrated CLL cells had significantly higher expression of CD49d (P = .02), matrix metallopeptidase-9 (P = .004), CD38 (P = .009), CD80 (P = .04), and CD69 (P = .04) compared with CLL cells that remained in the circulation. The degree of migration observed strongly correlated with CD49d expression (r(2), 0.47; P = .01), and treatment with the CD49d-blocking antibody natalizumab resulted in significantly decreased migration (P = .01). Taken together, our data provide evidence for a novel, dynamic, and tractable in vitro model of lymphocyte migration and confirm that CD49d is a critical regulator of this process in CLL.

CD49d is the strongest flow cytometry-based predictor of overall survival in chronic lymphocytic leukemia.

PURPOSE: Although CD49d is an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL), definitive validation evidence is lacking. A worldwide multicenter analysis was performed using published and unpublished CLL series to evaluate the impact of CD49d as an overall (OS) and treatment-free survival (TFS) predictor. PATIENTS AND METHODS: A training/validation strategy was chosen to find the optimal CD49d cutoff. The hazard ratio (HR) for death and treatment imposed by CD49d was estimated by pooled analysis of 2,972 CLLs; Cox analysis stratified by center and stage was used to adjust for confounding variables. The importance of CD49d over other flow cytometry-based prognosticators (eg, CD38, ZAP-70) was ranked by recursive partitioning. RESULTS: Patients with ≥ 30% of neoplastic cells expressing CD49d were considered CD49d+. Decrease in OS at 5 and 10 years among CD49d+ patients was 7% and 23% (decrease in TFS, 26% and 25%, respectively). Pooled HR of CD49d for OS was 2.5 (2.3 for TFS) in univariate analysis. This HR remained significant and of similar magnitude (HR, 2.0) in a Cox model adjusted for clinical and biologic prognosticators. Hierarchic trees including all patients or restricted to those with early-stage disease or those age ≤ 65 years always selected CD49d as the most important flow cytometry-based biomarker, with negligible additional prognostic information added by CD38 or ZAP-70. Consistently, by bivariate analysis, CD49d reliably identified patient subsets with poorer outcome independent of CD38 and ZAP-70. CONCLUSION: In this analysis of approximately 3,000 patients, CD49d emerged as the strongest flow cytometry-based predictor of OS and TFS in CLL.

A novel Cdk9 inhibitor preferentially targets tumor cells and synergizes with fludarabine.

Cdk9 is a key elongation factor for RNA transcription and functions by phosphorylating the C-terminal domain of RNA polymerase II. Here we present direct evidence that cdk9 is important for cancer cell survival and describe the characterization of the potent cdk9 inhibitor CDKI-73 in primary human leukemia cells. CDKI-73 induced caspase-dependent apoptosis that was preceded by dephosphorylation of cdk9 and serine 2 of RNA polymerase II. CDKI-73 was more potent than the pan-cdk inhibitor flavopiridol and showed >200-fold selectivity against primary leukemia cells when compared with normal CD34+ cells. Furthermore, CDKI-73 was equipotent in poor prognostic sub-groups of leukemia patients and showed cytotoxic synergy with the nucleoside analog fludarabine. The Mechanism of synergy was associated with CDKI-73-mediated transcriptional inhibition of MCL1 and XIAP that was maintained when used in combination with fludarabine. Our data present a strong rationale for the development of cdk9 inhibitors such as CDKI-73 as anticancer therapeutics.

A genome-wide association study identifies multiple susceptibility loci for chronic lymphocytic leukemia.

Genome-wide association studies (GWAS) of chronic lymphocytic leukemia (CLL) have shown that common genetic variation contributes to the heritable risk of CLL. To identify additional CLL susceptibility loci, we conducted a GWAS and performed a meta-analysis with a published GWAS totaling 1,739 individuals with CLL (cases) and 5,199 controls with validation in an additional 1,144 cases and 3,151 controls. A combined analysis identified new susceptibility loci mapping to 3q26.2 (rs10936599, P = 1.74 × 10(-9)), 4q26 (rs6858698, P = 3.07 × 10(-9)), 6q25.2 (IPCEF1, rs2236256, P = 1.50 × 10(-10)) and 7q31.33 (POT1, rs17246404, P = 3.40 × 10(-8)). Additionally, we identified a promising association at 5p15.33 (CLPTM1L, rs31490, P = 1.72 × 10(-7)) and validated recently reported putative associations at 5p15.33 (TERT, rs10069690, P = 1.12 × 10(-10)) and 8q22.3 (rs2511714, P = 2.90 × 10(-9)). These findings provide further insights into the genetic and biological basis of inherited genetic susceptibility to CLL.

Apoptosis deregulation in CLL.

The description of apoptosis and the identification of the genes that regulate it have proved pivotal to our understanding of how cancer cells accumulate and ultimately cause morbidity and mortality. It has become increasingly clear that in CLL the balance between the pro- and anti-apoptotic members of the BCL2 family of apoptotic regulatory proteins is critical in the development and clinical progression of CLL. Furthermore, the apoptotic potential of the CLL cell determines chemotherapy sensitivity and ultimately progression-free and overall survival. The unravelling of the BCL2 story in CLL has led to the development of a whole new class of therapeutic agents-the BH3 mimetics-which are significantly more targeted than conventional chemo-immunotherapy and therefore promise potent clinical activity coupled with reduced toxicity.