TP53 Mutations with Low Variant Allele Frequency Predict Short Survival in Chronic Lymphocytic Leukemia.

PURPOSE: In chronic lymphocytic leukemia (CLL), TP53 mutations are associated with reduced survival and resistance to standard chemoimmunotherapy (CIT). Nevertheless, the clinical impact of subclonal TP53 mutations below 10% to 15% variant allele frequency (VAF) remains unclear. EXPERIMENTAL DESIGN: Using a training/validation approach, we retrospectively analyzed the clinical and biological features of TP53 mutations above (high-VAF) or below (low-VAF) the previously reported 10.0% VAF threshold, as determined by deep next-generation sequencing. Clinical impact of low-VAF TP53 mutations was also confirmed in a cohort (n = 251) of CLL treated with fludarabine-cyclophosphamide-rituximab (FCR) or FCR-like regimens from two UK trials. RESULTS: In the training cohort, 97 of 684 patients bore 152 TP53 mutations, while in the validation cohort, 71 of 536 patients had 109 TP53 mutations. In both cohorts, patients with the TP53 mutation experienced significantly shorter overall survival (OS) than TP53 wild-type patients, regardless of the TP53 mutation VAF. By combining TP53 mutation and 17p13.1 deletion (del17p) data in the total cohort (n = 1,220), 113 cases were TP53 mutated only (73/113 with low-VAF mutations), 55 del17p/TP53 mutated (3/55 with low-VAF mutations), 20 del17p only, and 1,032 (84.6%) TP53 wild-type. A model including low-VAF cases outperformed the canonical model, which considered only high-VAF cases (c-indices 0.643 vs. 0.603, P < 0.0001), and improved the prognostic risk stratification of CLL International Prognostic Index. Clinical results were confirmed in CIT-treated cases (n = 552) from the retrospective cohort, and the UK trials cohort. CONCLUSIONS: TP53 mutations affected OS regardless of VAF. This finding can be used to update the definition of TP53 mutated CLL for clinical purposes.

CD49d promotes disease progression in chronic lymphocytic leukemia: new insights from CD49d bimodal expression.

CD49d is a remarkable prognostic biomarker of chronic lymphocytic leukemia (CLL). The cutoff value for the extensively validated 30% of positive CLL cells is able to separate CLL patients into 2 subgroups with different prognoses, but it does not consider the pattern of CD49d expression. In the present study, we analyzed a cohort of 1630 CLL samples and identified the presence of ∼20% of CLL cases (n = 313) characterized by a bimodal expression of CD49d, that is, concomitant presence of a CD49d+ subpopulation and a CD49d- subpopulation. At variance with the highly stable CD49d expression observed in CLL patients with a homogeneous pattern of CD49d expression, CD49d bimodal CLL showed a higher level of variability in sequential samples, and an increase in the CD49d+ subpopulation over time after therapy. The CD49d+ subpopulation from CD49d bimodal CLL displayed higher levels of proliferation compared with the CD49d- cells; and was more highly represented in the bone marrow compared with peripheral blood (PB), and in PB CLL subsets expressing the CXCR4dim/CD5bright phenotype, known to be enriched in proliferative cells. From a clinical standpoint, CLL patients with CD49d bimodal expression, regardless of whether the CD49d+ subpopulation exceeded the 30% cutoff or not, experienced clinical behavior similar to CD49d+ CLL, both in chemoimmunotherapy (n = 1522) and in ibrutinib (n = 158) settings. Altogether, these results suggest that CD49d can drive disease progression in CLL, and that the pattern of CD49d expression should also be considered to improve the prognostic impact of this biomarker in CLL.

Cluster analysis of immunophenotypic data: the example of chronic lymphocytic leukemia.

Studies of gene expression profiling have been successfully used for the identification of molecules to be employed as potential prognosticators. In analogy with gene expression profiling, we have previously proposed an original method to identify the immunophenotypic signature of chronic lymphocytic leukemia (CLL) subsets with different prognosis, named surface-antigen expression profiling. According to this method, expression data for surface markers can be successfully analyzed by data mining tools identical to those employed in gene expression profiling studies, including unsupervised and supervised algorithms, with the aim to identify the immunophenotypic signature of CLL subsets with different prognosis. By employing an identical approach for investigating the reactivity of a wide panel of monoclonal antibodies provided by the "Ninth International Workshop on Leukocyte Differentiation Antigens", we were able to identify some of them (i.e. TCL1, CCR7, FCRL2, FCRL3, and CD150) as additional potential markers with prognostic relevance in CLL. These suggestions need to be confirmed: (i) in a new set of clinically characterized CLL cases; (ii) in combination with other prognostic markers in the context of comprehensive scoring systems for clinical outcome prediction.

An interactive diary for diet management (DAI): a new telemedicine system able to promote body weight reduction, nutritional education, and consumption of fresh local produce.

BACKGROUND: The aim of this multicenter, longitudinal, single-arm, pre-post comparison was to test a telemedicine system able to promote body weight reduction, nutritional education, and consumption of fresh local produce. METHODS: DAI (MeTeDa srl, San Benedetto del Tronto, Italy) is a software for mobile phones to support patients following a specific dietetic program. It facilitates the communication between the patient and dietician via short text messages. Overall, three specialized dieticians enrolled 140 consecutive patients with body mass index (BMI) >or=25 kg/m(2) who voluntered to follow a specific diet program to be managed with DAI. At baseline and after 20 weeks, data on body weight, waist circumference, BMI, fasting blood glucose, lipid profile, food habits, and physical activity were collected and compared by the Wilcoxon test or the McNemar test. RESULTS: Overall, 115 individuals (82.1%) completed the follow-up. The mean (95% confidence interval) reduction in body weight was -2.5 (-3.2; -1.8) kg, whereas the reduction in waist circumference was -3.7 (-4.6; -2.9) cm, and that in BMI was 1.0 (-0.7; -1.2) kg/m(2). The software was useful as an educational tool: participants achieving the Mediterranean diet targets increased from 14.4% to 69.8% after 20 weeks. On average, each patient recognized and chose fresh local vegetables eight times per week during the follow-up. Participants regularly communicated with dieticians through short text messages. CONCLUSIONS: This study allowed the documentation of the efficacy of a new telemedicine system in supporting people who need to lose body weight. The tool was also suitable for a more articulated initiative of "nutritional education" aiming to promote the healthy properties of the Mediterranean diet and the consumption of local produce.

ZAP-70 expression in B-cell chronic lymphocytic leukemia: evaluation by external (isotypic) or internal (T/NK cells) controls and correlation with IgV(H) mutations.

BACKGROUND: Expression of T cell specific zeta-associated protein 70 (ZAP-70) by B-cell chronic lymphocytic leukemia (B-CLL) cells, as investigated by flow cytometry, has both prognostic relevance and predictive power as surrogate for immunoglobulin heavy chain variable region (IgV(H)) mutations, although a standardization of the cytometric protocol is still lacking. METHODS: Flow cytometric analyses for ZAP-70 were performed in peripheral blood samples from 145 B-CLL (124 with IgV(H) mutations) by a standard three-color protocol. Identification of ZAP-70(+) cell population was based on an external negative control, i.e., the isotypic control (ISO method) or an internal positive control, i.e., the population of residual normal T/NK cells (TNK method). A comparison between these two approaches was performed. RESULTS: While 86/145 cases were concordant as for ZAP-70 expression according to the two methods (ISO(+)TNK(+) or ISO(-)TNK(-)), 59/145 cases had discordant ZAP-70 expression, mainly (56/59) showing a ISO(+)TNK(-) profile. These latter cases express higher levels of ZAP-70 in their normal T cell component. Moreover, discordant ISO(+)TNK(-) cases had a IgV(H) gene mutation profile similar to that of concordantly positive cases and different from ZAP-70 concordantly negative B-CLL. CONCLUSION: Analysis of ZAP-70 expression by B-CLL cells by using the ISO method allows to overcome the variability in the expression of ZAP-70 by residual T cells and yields a better correlation with IgV(H) gene mutations. A receiver operating characteristic analysis suggests to employ a higher cut-off than the commonly used 20%. A parallel evaluation of the prognostic value of ZAP-70 expression, as determined according to the ISO and TNK methods, is still needed.

A scoring system based on the expression of six surface molecules allows the identification of three prognostic risk groups in B-cell chronic lymphocytic leukemia.

We have previously identified 12 surface antigens whose differential expression represented the signature of B-cell chronic lymphocytic leukemia (B-CLL) subsets with different prognosis. In the present study, expression data for these antigens, as determined in 137 B-CLL cases, all with survivals, were utilized to devise a comprehensive immunophenotypic scoring system of prognostic relevance for B-CLL patients. In particular, univariate z score was employed to identify the markers with greater prognostic impact, while maximally selected log-rank statistics were chosen to define the optimal cut-off points capable to split patients into two groups with different survivals. A weighted immunophenotypic scoring system was developed by integrating results from these analyses. Six antigens were selected: three positive prognosticators (CD62L, CD54, CD49c) and three negative prognosticators (CD49d, CD38, CD79b), with cut-off values ranging from 30% to 50% of positive cells. By weighing the expression of each marker according to its statistical power, a complete scoring system, with point values comprised between 0 (complete absence of phenotypic conditions associated with good prognosis) and 9 (all the phenotypic conditions associated with good prognosis fulfilled), allowed to split the whole set of B-CLL patients, into three distinctive prognostic groups (P = 4.78 x 10(-11)) with high- (score 0-3), intermediate- (score 4-6), and low- (score 7-9) risk of death. The three risk groups showed different distribution of cases as for Rai's stages, IgVH mutations, and ZAP-70 expression. The proposed immunophenotypic scoring system may be an additional useful tool in routine diagnostic/prognostic procedures for B-CLL.

Methylation-regulated expression of cancer testis antigens in primary effusion lymphoma: immunotherapeutic implications.

Primary effusion lymphoma (PEL) is a large B-cell neoplasm with an unfavorable prognosis and limited therapeutic options. In this study, cancer testis antigens (CTA) were investigated as potential immunotherapeutic targets in patients with PEL. Baseline expression of a panel of 11 CTA was highly heterogeneous among five PEL cell lines. In particular, the investigated CTA were not expressed in BC-2 and BC-3 cells, while BC-1, HBL-6, and BCBL-1 cells tested positive for 6, 8, and 9 CTA, respectively. The DNA hypomethylating agent 5-aza-2'-deoxycytdine (5-AZA-CdR) invariably induced or up-regulated the expression of all investigated CTA in all cell lines analyzed. The de novo expression of CTA was still detectable at mRNA and protein level at least 2 months after the end of 5-AZA-CdR treatment. These findings, and the concomitant up-regulation of HLA-class I antigens and ICAM-1 by 5-AZA-CdR, support its clinical use to set-up innovative chemo-immunotherapeutic approaches in PEL.

Mutational status of IgVH genes consistent with antigen-driven selection but not percent of mutations has prognostic impact in B-cell chronic lymphocytic leukemia.

Mutational status of immunoglobulin heavy-chain variable-region (IgVH) genes, along with CD38 expression, is a prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL). Configuration of IgVH genes displaying > 2% mismatch has been shown to correlate with longer survivals. In a series of 64 B-CLLs, we failed to confirm the prognostic value of the IgVH gene mutational status by using the suggested cutoff. However, the IgVH mutational status maintained its prognostic value only when evidence of antigen-driven selection could be documented. This was accomplished by applying statistical methods aimed at evaluating a significant skewing of replacement mutations from framework to complementary determining regions, as it occurs during germinal center differentiation of B cells. These data caution against wide application of the 2% somatic mutation cutoff as a prognostic determinant without demonstration of antigen-driven selection.

Analysis of IgV gene mutations in B cell chronic lymphocytic leukaemia according to antigen-driven selection identifies subgroups with different prognosis and usage of the canonical somatic hypermutation machinery.

Cases of B-cell chronic lymphocytic leukaemia (B-CLL) with mutated (M) IgV(H) genes have a better prognosis than unmutated (UM) cases. We analysed the IgV(H) mutational status of B-CLL according to the features of a canonical somatic hypermutation (SHM) process, correlating this data with survival. In a series of 141 B-CLLs, 124 cases were examined for IgV(H) gene per cent mutations and skewing of replacement/silent mutations in the framework/complementarity-determining regions as evidence of antigen-driven selection; this identified three B-CLL subsets: significantly mutated (sM), with evidence of antigen-driven selection, not significantly mutated (nsM) and UM, without such evidence and IgV(H) gene per cent mutations above or below the 2% cut-off. sM B-CLL patients had longer survival within the good prognosis subgroup that had more than 2% mutations of IgV(H) genes. sM, nsM and UM B-CLL were also characterized for the biased usage of IgV(H) families, intraclonal IgV(H) gene diversification, preference of mutations to target-specific nucleotides or hotspots, and for the expression of enzymes involved in SHM (translesion DNA polymerase zeta and eta and activation-induced cytidine deaminase). These findings indicate the activation of a canonical SHM process in nsM and sM B-CLLs and underscore the role of the antigen in defining the specific clinical and biological features of B-CLL.

CD26 expression correlates with a reduced sensitivity to 2'-deoxycoformycin-induced growth inhibition and apoptosis in T-cell leukemia/lymphomas.

PURPOSE AND EXPERIMENTAL DESIGN: dCF (2'-deoxycoformycin) is a potent inhibitor of ADA (adenosine deaminase), an enzyme regulating intra- and extracellular concentrations of purine metabolites. ADA exists as cytosolic and extracellular forms, the latter colocalized on the cell surface with CD26. Once the surface expression of CD26 and ADA in a panel of cell lines and primary samples of T-cell leukemia/lymphoma was defined, we correlated this expression with the antiproliferative and apoptotic effect of dCF. RESULTS: Surface expression of CD26 inversely correlated with the capability of dCF to inhibit cell growth and induce apoptosis both in T-cell lines and primary samples of T-cell malignancies. This conclusion was sustained by a decreased sensitivity to dCF-mediated proapoptotic and/or antiproliferative in vitro effects of: (a) leukemia/lymphoma T-cell lines expressing surface CD26/ADA complex; (b) primary CD26(+) T cell malignancies; and (c) normal T cells (CD26(+)) as compared with tumor T cells (CD26(-)) in unpurified samples from three cases of T-cell receptor gammadelta(+) T-cell malignancies characterized by a mixture of normal and neoplastic cells. This latter point was confirmed in vivo, in a patient affected by CD26(-) T-cell receptor gammadelta(+) hepatosplenic gammadelta(+) T-cell lymphomas treated on a compassionate basis with dCF. The inverse correlation between CD26 expression and sensitivity to dCF was also demonstrated in a lymphoblastic lymphoma case in which CD26 was expressed on circulating blasts at relapse but not at diagnosis, as well as in two H9 T-cell clones expressing or not expressing CD26 mRNA and protein. CONCLUSIONS: This study corroborates the notion of CD26 as a marker of poor prognosis for T-cell malignancies and delineates a role for CD26 as a predictor of poor response to dCF.

CD40L induces proliferation, self-renewal, rescue from apoptosis, and production of cytokines by CD40-expressing AML blasts.

OBJECTIVE: Conflicting experimental and clinical results have been reported regarding the role of CD40 in acute myeloid leukemia (AML). In the present study, we analyzed the capability of CD40L/CD154 to modulate several functional aspects of CD40-expressing AML blasts. METHODS: After defining the constitutive expression levels of CD40 in a wide panel (n = 67) of AMLs and evaluating the capability of cytokines to modulate its expression, we investigated the effects of CD40 engagement by soluble (s) CD40L on proliferation, self-renewal capacity, apoptosis, homotypic adhesion, and cytokine production of leukemia cells. RESULTS: CD40 was detected in blast cells from about 37% of AMLs, the highest frequency being documented in monocytic subtypes, and its expression was upregulated or de novo induced by treatment with interleukin (IL)-1alpha, IL-3, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma, and tumor necrosis factor-alpha. Exposure of CD40(+) AML blasts to sCD40L resulted in a dose-dependent proliferative response, enhancement of clonogenic growth and self-renewal capacity, and a striking increase in colony size. CD40 engagement was able to rescue AML blasts from apoptosis induced by serum deprivation, as demonstrated by reduced expression of APO2.7 and annexin-V binding, as well as upregulation of the anti-apoptotic protein bcl-x(L). CD40 triggering upregulated cell surface expression of the adhesion molecules CD54, CD58, and CD15 and resulted in homotypic aggregation of leukemia cells at least in part CD54-dependent. An increased production of IL-6 and GM-CSF by CD40(+) AML blasts was also documented upon sCD40L exposure. CONCLUSIONS: This study indicates a possible involvement of CD40 in the interactions of AML blasts with other growth-sustaining microenvironmental accessory cells and immune effectors, in turn expressing CD40L. Caution in the use of CD40 triggering in immunotherapy of AMLs is also suggested.

Hodgkin and Reed-Sternberg cells express functional c-kit receptors and interact with primary fibroblasts from Hodgkin's disease-involved lymph nodes through soluble and membrane-bound stem cell factor.

Classic Hodgkin's disease (cHD) is a lymphoid neoplasia characterized by few malignant Hodgkin and Reed-Sternberg (H-RS) cells, embedded in an abundant background of non-tumour cells. We have previously demonstrated the expression in primary H-RS cells of the receptor tyrosine kinase (RTK) c-kit; here we describe its functional role in the cross-talk between H-RS cells themselves with neighbouring cell populations. In particular, we analysed the expression of c-kit and its ligand stem cell factor (SCF) in a panel of HD-derived cell lines and fibroblasts from HD-involved lymph nodes (HDF). While c-kit was expressed by HD-derived cell lines, usually in the absence of SCF, this latter molecule, in its soluble and/or membrane-bound (mb) form, was in turn expressed at a high level by primary HDF. In vitro adhesion between HD-derived cell lines and HDF was mainly mediated by c-kit/SCF interactions, and this phenomenon was significantly inhibited by an excess of soluble SCF or by neutralizing anti-c-kit monoclonal antibodies. Furthermore, both soluble and mb-SCF increased growth and colony survival of HD-derived cell lines; these effects were significantly enhanced upon co-stimulation of H-RS cells with interleukin 9. Finally, soluble SCF was able to partially rescue H-RS cells from apoptosis induced by serum starvation. Taken together, our data indicated the expression of functional c-kit receptor by H-RS cells and suggests a role of SCF in the pathobiology of cHD.

Expression of functional interleukin-3 receptors on Hodgkin and Reed-Sternberg cells.

The human interleukin-3 receptor (IL-3R) is a heterodimeric complex consisting of an IL-3-specific alpha chain (IL-3Ralpha) and a common beta chain (beta(c)), this latter shared with the receptors for granulocyte-macrophage colony-stimulating factor and IL-5. Despite extensive research on cytokine circuitries regulating proliferation and survival of tumor cells in Hodgkin's disease (HD) the functional expression of IL-3Rs in this pathobiological entity has not yet been investigated. In the present study, we demonstrate that the great majority (>90%) of malignant Hodgkin and Reed-Sternberg cells of classic HD (19 of 19 analyzed cases) express IL-3Ralpha by immunostaining of frozen sections and cell suspensions from involved lymph nodes. Accordingly, HD cell lines (L428, KMH2, HDLM2, L1236) expressed the alpha and beta chains of IL-3R both at the mRNA and protein level, with a molecular size of IL-3Ralpha identical (70 kd) to that expressed by human myeloid cells. Exogenous IL-3 promoted the growth of cultured Hodgkin and Reed-Sternberg cells, such effect being potentiated by IL-9 co-stimulation, and was able to partially rescue tumor cells from apoptosis induced by serum deprivation. This data suggests an involvement of IL-3/IL-3R interactions in the cellular growth of HD through paracrine mechanisms.