Extracellular vesicles (EV) are membranous particles (30–1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer) and MEC1 (B‐cell chronic lymphocytic leukaemia; CLL), were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19⁺ EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA‐A,B,C, HLA‐DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age‐ and gender‐matched healthy individuals.

Many prognostic markers have been identified in chronic lymphocytic leukemia, but there have been few opportunities to assess their relative importance in a large randomized trial. The aim of this study was to determine which of the available markers independently predicted outcome in patients requiring treatment and to use these to define new risk groups. A broad panel of clinical and laboratory markers, measured at randomization in patients entering the LRF CLL4 trial, was assessed with respect to treatment response, progression-free and overall survival, at a median follow-up of 68 months. Using the factors identified as independent predictors for progression-free survival, patients were subdivided into three risk groups: 6% had poor risk with known TP53 loss of greater than 10%; 72% had an intermediate risk without TP53 loss (≤ 10%) and with at least one of: unmutated IGHV genes and/or IGHV3-21 usage, 11q deletion, β-2 microglobulin greater than 4 mg/L; 22% had a good risk (with none of the above and mutated IGHV genes). The 5-year progression-free survival rates for these three groups were 0%, 12% and 34%, respectively, and the corresponding 5-year overall survival rates were 9%, 53% and 79% (both P<0.00005 independent of treatment allocation). In the intermediate risk group 250 patients, with data for all three risk factors, were further subdivided into intermediate-low (one risk factor) or intermediate-high (2 or 3 risk factors). The 5-year progression-free survival rates were 18% and 7% (P=0.0001) and the 5-year overall survival rates were 68% and 40% (P<0.00005), respectively. This study demonstrates the role of biomarkers in prognosis and shows that, in patients requiring treatment, disease stage may no longer be an independent predictor of outcome. If validated independently, the risk groups defined here may inform the design of future trials in chronic lymphocytic leukemia.

Hypogammaglobulinemia is a common complication of chronic lymphocytic leukemia (CLL), but the significance of immunoglobulin G (IgG) subclass deficiency is unknown. We analyzed the prevalence of immunoglobulins G, A and M, IgG subclass deficiency and infection in 150 patients with CLL. Low IgG, IgA and IgM levels were observed in 27.3%, 30.7% and 56.7% of patients, respectively. IgG subclass deficiency was frequent, with reduced IgG1, IgG2, IgG3 and IgG4 in 28%, 19.3%, 52% and 22.7% of patients, respectively. IgG subclass deficiency (total 64.6%) and hypogammaglobulinemia (27.3%) were more prevalent than clinically significant infection (16%). Recurrent or significant infections were seen in 24 patients (16%), of whom 50% had hypogammaglobulinemia but 100% had at least one IgG subclass deficiency, indicating that half the patients with infection had IgG subclass deficiency but normal total IgG level. Deficiencies of IgG3 and IgG4 were statistically associated with infection risk. Normal immunoglobulin and IgG subclass levels were seen in 26 patients (17%) and none had infections. IgG subclass deficiency is commonly observed in patients with CLL with both normal and reduced total IgG levels, and is associated with infection. Screening patients with CLL for IgG subclass deficiency may be a useful adjunct in stratifying their infection risk.

Associated with low cellular drug resistance to asparaginase, cytarabine and corticosteroids. These data might help to understand the effect of different treatment regimens and determine the most efficient therapy for this group of patients.

Numerous studies in chronic lymphocytic leukaemia (CLL) have demonstrated the adverse prognostic significance of structural abnormalities of chromosomes 17p13 and 11q23, regions that encompass the TP53 and ATM genes, respectively. The identification of patients with progressive disease and a TP53 abnormality is particularly important as most are resistant to purine analogues and alkylating agents but frequently respond to alemtuzumab, which kills by a TP53-independent mechanism. Most studies, including recent large randomized trials of initial therapy for CLL, utilize interphase fluorescence in situ hybridization to detect TP53 and ATM loss. The cutoff level for TP53 loss that correlates best with clinical outcome remains controversial. Many studies have used a 3–5% cutoff based on the level of aberrant signal patterns to be expected in normal controls. However, in the UK CLL4 trial, only cases with >20% TP53 loss had a poor response to initial therapy and short progression free and overall survival.1 TP53 loss is frequently accompanied by a mutation of the remaining allele.2 The mutations are usually missense mutations and the mutant protein has a prolonged half-life enabling its detection by flow cytometry, western blotting or immunostaining. Less than 5% of patients have a TP53 mutation without loss of the normal allele and would not be identified if fluorescence in situ hybridization was used as the sole screening test. The single large study in which unselected CLL patients were screened for the presence of ATM mutations found an incidence of 12%.3 A follow-up study identified ATM mutations with deletion of the ATM gene in only 38% of patients, contrasting with the high concordance found between TP53 loss and mutation. Preliminary data suggest that the outcome of patients with an ATM mutation is poorer than for those with ATM loss without mutation.4 Both the TP53 and ATM genes are integral to the DNA damage response pathway to double-stranded DNA breaks. Functional assays of this pathway, measuring activation of TP53 and ATM, upregulation of downstream genes or resistance to apoptosis, have been developed and have potential diagnostic and prognostic value. Carter et al.5 measured expression of TP53 and p21, initially by western blotting, and more recently by flow cytometry before and following exposure of CLL cells to ionizing irradiation. TP53 dysfunction was defined as a failure to upregulate p21 expression following irradiation. Cases could be subdivided into those with increased basal levels of TP53 (type A) and those with low baseline TP53 expression (type B). In the initial study, there was a perfect correlation between type A dysfunction and the presence of a TP53 mutation, and type B dysfunction and the presence of an ATM mutation. However, a recent update while confirming the strong association between type A dysfunction and TP53 loss in greater than 50% of cells detected type B dysfunction in cases with TP53, ATM or 13q loss.6 Nutlin-3a, a small-molecule inhibitor of the interaction between TP53 and MDM2, upregulates TP53 and p21 expression in cells with wild-type TP53, and can induce apoptosis alone or in combination with alkylating agents, purine analogues or topoisomerase 2 inhibitors in CLL cells in vitro. Increased apoptosis has been demonstrated in small numbers of cases with a deletion of 11q or low ATM protein levels, but not in those with a TP53 mutation. The aim of this study was to develop a functional assay suitable for routine clinical use, capable of identifying and distinguishing abnormalities of TP53 and ATM. To achieve this, we employed a radiomimetic drug etoposide, which obviated the need for a radiation source, used alone and in combination with nutlin-3a. Forty-seven patients were selected based on a prior screen for the presence of TP53 and ATM loss by fluorescence in situ hybridization and for mutations using direct sequencing of exons 2–9 and denaturing high-performance liquid chromatography, respectively. Twelve had no detectable TP53 or ATM abnormality, 15 had TP53 loss and/or mutation and 20 had ATM loss and/or mutation (Table 1). Cryopreserved mononuclear cells from each case were thawed and exposed to 50 M etoposide alone, or 50 M etoposide plus 5 M nutlin-3a. Initially, we defined cutoff values using receiver operating characteristic analysis for the expression of TP53 and p21 following etoposide treatment. Percentage increases in TP53 and p21 expression below 30 and 15% respectively defined dysfunctional cases. Those cases in which function was restored with the combination of etoposide and nutlin-3a were classified as category 2 dysfunction, while failure to restore function was classified as category 1. The combination of etoposide and nutlin-3a was better at distinguishing category 1 from category 2 dysfunction than nutlin-3a alone. In addition, we confirmed that cryopreservation did not affect functional categorization by comparing data on fresh and frozen cells from representative cases in each functional category. The responses of representative cases from each functional category are shown in Figure 1a. No dysfunction was detected in the 12 cases with no TP53 or ATM abnormality. Category 1 dysfunction was found in all 11 patients with a TP53 mutation, including 3 without TP53 loss. Category 2 dysfunction occurred in all 16 cases with an ATM mutation including 4 without ATM loss. No dysfunction was detected in the four cases with TP53 loss or eight cases of ATM loss lacking a mutation of the remaining allele (Table 1). Factors that could potentially affect the sensitivity of the functional assay include the ability of etoposide to reliably induce double-stranded DNA breaks, especially in drug-resistant cases, the choice of TP53 and p21 expression as markers of TP53 dysfunction, the size of the mutant and deleted clones and the nature of the TP53 or ATM mutations. A comparison of etoposide and -irradiation showed no...