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Antonella Zucchetto - One of the best experts on this subject based on the ideXlab platform.

  • CD49d prevails over the novel recurrent mutations as independent prognosticator of overall survival in chronic lymphocytic leukemia
    Leukemia, 2016
    Co-Authors: M. Dal Bo, Antonella Zucchetto, D Benedetti, Riccardo Bomben, E Tissino, P Bulian, F M Rossi, F Pozzo, T Bittolo, P Nanni
    Abstract:

    CD49d, the alpha-chain of the integrin heterodimer α4β1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53 . In addition to TP53 , the clinical relevance of NOTCH1 , SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53 , NOTCH1 , SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P< 0.0001). Consistently, high CD49d expression identified CLL subsets with inferior OS in the context of each category of a previously reported hierarchical risk stratification model. Moreover, by evaluating the relative importance of biological prognosticators by random survival forests, CD49d was selected among the top-ranked OS predictor (variable importance =0.0410), along with IGHV mutational status and TP53 abnormalities. These results confirmed CD49d as an independent negative OS prognosticator in CLL also in comprehensive models comprising the novel recurrent mutations. In this context, TP53 disruption and NOTCH1 mutations retained prognostic relevance, in keeping with their roles in CLL cell immuno-chemoresistance.

  • Functional and clinical significance of the integrin alpha chain CD49d expression in chronic lymphocytic leukemia
    Current cancer drug targets, 2016
    Co-Authors: Michele Dal Bo, Riccardo Bomben, Francesca Rossi, Giovanni Del Poeta, Dania Benedetti, Chiara Caldana, Erika Tissino, Gianluca Gaidano, Pietro Bulian, Antonella Zucchetto
    Abstract:

    Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation/expansion of a clonal population of neoplastic cells with the morphological appearance of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. CD49d, the α chain of the α4β1 integrin heterodimer, is one of the main interactors between CLL cells and accessory cells in the microenvironmental sites and one of the main predictors of overall survival. In particular, CD49d is known to play a pivotal role in mediating both cell-cell and cell-matrix interactions in CLL-involved tissues eventually delivering prosurvival signals and protecting CLL cells from drug-induced damages. Treatment strategies targeting the α4β1 integrin could represent an interesting option in CLL. In this context, the recombinant anti-CD49d antibody natalizumab demonstrated the potential to overcome stromal cell-induced resistance of B cell lymphoma cells against cytotoxic drugs and rituximab in vitro. Moreover, a specific interest for the CD49d molecule raises from the clinical activity of the recently proposed inhibitors of kinases downstream the BCR that has been recently related with the inside-out activation of the α4β1 integrin. In the review, we addressed in detail the role of CD49d in CLL cells, including clinical impact, relationship with specific cytogenetic features, and CD49d-dependent interactions in lymph node and bone marrow microenvironment responsible for growth- and survival- supporting signals, eventually influencing CLL prognosis and therapeutic options.

  • NOTCH1 Mutations Are Associated with High CD49d Expression in Chronic Lymphocytic Leukemia
    Blood, 2014
    Co-Authors: Antonella Zucchetto, Riccardo Bomben, Francesca Rossi, Dania Benedetti, Chiara Caldana, Erika Tissino, Federico Pozzo, Silvia Rasi, Carmela Ciardullo, Francesco Zaja
    Abstract:

    Mutations of NOTCH1 have emerged as one of the most frequent somatic alterations in chronic lymphocytic leukemia (CLL), affecting up to 10–15% of patients. These mutations (~80% are 7544-7545delCT frameshift deletions) generate a truncated protein that accumulates in the cell and activates the downstream NOTCH1 signaling which is implicated in apoptosis resistance and increased survival of CLL cells. CD49d (α4 integrin chain) is one of the most relevant negative prognosticator in CLL, expressed by ~40% of CLL cases, and associated with aggressive/accelerated clinical courses, whose key role in CLL cell microenvironmental interactions has been thoroughly investigated. Literature data indicate that NOTCH1 has a role in activating the integrin signaling in several cell models. Given the higher CD49d expression characterizing trisomy 12 CLL, a CLL subset in which the NOTCH1 pathway is more often activated by NOTCH1 mutations, this study was aimed at investigating the contribution of NOTCH1 in the regulation of CD49d expression in CLL. The 7544-7545delCT NOTCH1 mutations were investigated by ARMS-PCR in 1027 CLL cases, all characterized for CD49d expression and for the cytogenetic profiles by FISH. NOTCH1 mutated cases were 158/1027 (15%), with a higher prevalence (36.7% of NOTCH1 mutated cases) in the trisomy 12 cytogenetic group. Analysis of CD49d expression highlighted a very strong association between the presence of NOTCH1 mutations and CD49d expression (p 30% of positive cells) was found in 102/158 (64.5%) NOTCH1 mutated cases as compared to 285/869 (32.8%) NOTCH1 wild-type cases. Of note, excluding trisomy 12 CLL, again NOTCH1 mutated CLL (100/865, 11.6%) displayed a significantly higher frequency of CD49d+ cases (52%) as compared to NOTCH1 wild-type CLL (25.7%) (p We next analyzed the percentage of mutated NOTCH1 DNA in the context of the CLL clone by a quantitative real-time PCR (QRT-PCR) approach set up by us to quantify the delCT NOTCH1 mutation. Using the 10% cut-off value to discriminate between cases with high (high NOTCH1) and low (low NOTCH1) mutation burden, 90/138 (67.7%) and 43/138 (32.3%) CLL cases were classified as low NOTCH1 and high NOTCH1, respectively. A higher prevalence of CD49d+ cases was found in the high NOTCH1 group (79%) as compared to the low NOTCH1 group (58%) (p=0.03). Moreover, a significant association between CD49d expression and a high NOTCH1 mutation burden was observed also excluding trisomy 12 CLL, with 69% of CD49d+ cases in the high NOTCH1 group, compared to 41% of CD49d+cases in the low NOTCH1 group (p=0.03). The association between NOTCH1 mutations and CD49d expression was next confirmed by next-generation sequencing results using the flow cytometrically sorted (>99% purity) CD49d- and CD49d+ components from 8 CLL cases characterized by both CD49d bimodal expression, and the presence of 7544-7545delCT NOTCH1 mutations at the subclonal level. In 7/8 cases, the CD49d+ component showed a higher NOTCH1 mutation burden compared to the CD49d- component, this difference reaching statistical significance in 4/7 cases. Of note, a similar clustering of mutations could not be observed in the CD49d- and CD49d+ components of other CLL cases characterized by bimodal CD49d expression and subclonal mutations of SF3B1 (n=1), BIRC3 (n=2) or TP53 (n=2). To verify whether NOTCH1 accumulation, as occurring in NOTCH1 mutated CLL, may influence CD49d expression, MEC-1 cells were transfected with a vector containing either a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or a NICD carrying a missense mutation (c.5304G>A) generating a stop codon at the beginning of the sequence, as control. The higher levels of both NOTCH1 transcript (fold increase over control=2.2) and protein (fold increase over control=1.3) characterizing mutated-NICD MEC-1 cells, was paralleled by higher levels of CD49d expression (mean fluorescence intensity=23.300 versus 12.400) in these cells. Altogether our data demonstrate a direct correlation between NOTCH1 mutations and CD49d expression also outside the trisomy 12 CLL group, and suggest that accumulation of NOTCH1 may be directly or indirectly responsible for the up-regulation of CD49d expression. Disclosures No relevant conflicts of interest to declare.

  • CD49d is the strongest flow cytometry based predictor of overall survival in chronic lymphocytic leukemia
    Journal of Clinical Oncology, 2014
    Co-Authors: Pietro Ulia, Antonella Zucchetto, Tai D Shanafel, Christophe Fega, Lilla Cro, Holge Nuckel, Luca Aldini, Antonina V Kurtova, Alessandra Ferrajoli, Ja A Urge
    Abstract:

    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...

  • CD49d is the strongest flow cytometry based predictor of overall survival in chronic lymphocytic leukemia
    Blood, 2013
    Co-Authors: Pietro Ulia, Antonella Zucchetto, Tai D Shanafel, Christophe Fega, Lilla Cro, Holge Nuckel, Luca Aldini, Antonina V Kurtova, Alessandra Ferrajoli, Ja A Urge
    Abstract:

    ![Graphic][1] Purpose Although CD49d is an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL), definitive validation evidence is lacking. A worldwide multi-center analysis was performed using published and unpublished CLL series to evaluate the impact of CD49d as overall survival (OS) and treatment free survival (TFS) predictor. Patients and Methods A training/validation strategy was chosen to find the optimal CD49d cut-off. The hazard ratio (HR) for death and treatment imposed by CD49d was estimated by pooled analysis of 2,972 CLL, and Cox analysis stratified by center and stage was used to adjust for confounding variables. The importance of CD49d over other flow cytometry-based prognosticators (CD38, ZAP-70) was ranked by recursive partitioning. Results Patients with ≥30% of neoplastic cells expressing CD49d were considered CD49d+. The decrease in OS at 5 and 10-years among CD49d+ cases was 7% and 23% (decrease in TFS 26% and 25% respectively). The 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 biological prognosticators. Hierarchical trees including all cases, or restricted to early stage or patients ≤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 patients' subsets with poorer outcome independent of CD38 and ZAP-70. Conclusions In this analysis of ∼3000 patients, CD49d emerged as the strongest flow cytometry-based predictor of OS and TFS in CLL. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Burger: Pharmacyclics: Research Funding. [1]: /embed/inline-graphic-2.gif

Francesca Rossi - One of the best experts on this subject based on the ideXlab platform.

  • Functional and clinical significance of the integrin alpha chain CD49d expression in chronic lymphocytic leukemia
    Current cancer drug targets, 2016
    Co-Authors: Michele Dal Bo, Riccardo Bomben, Francesca Rossi, Giovanni Del Poeta, Dania Benedetti, Chiara Caldana, Erika Tissino, Gianluca Gaidano, Pietro Bulian, Antonella Zucchetto
    Abstract:

    Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation/expansion of a clonal population of neoplastic cells with the morphological appearance of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. CD49d, the α chain of the α4β1 integrin heterodimer, is one of the main interactors between CLL cells and accessory cells in the microenvironmental sites and one of the main predictors of overall survival. In particular, CD49d is known to play a pivotal role in mediating both cell-cell and cell-matrix interactions in CLL-involved tissues eventually delivering prosurvival signals and protecting CLL cells from drug-induced damages. Treatment strategies targeting the α4β1 integrin could represent an interesting option in CLL. In this context, the recombinant anti-CD49d antibody natalizumab demonstrated the potential to overcome stromal cell-induced resistance of B cell lymphoma cells against cytotoxic drugs and rituximab in vitro. Moreover, a specific interest for the CD49d molecule raises from the clinical activity of the recently proposed inhibitors of kinases downstream the BCR that has been recently related with the inside-out activation of the α4β1 integrin. In the review, we addressed in detail the role of CD49d in CLL cells, including clinical impact, relationship with specific cytogenetic features, and CD49d-dependent interactions in lymph node and bone marrow microenvironment responsible for growth- and survival- supporting signals, eventually influencing CLL prognosis and therapeutic options.

  • NOTCH1 Mutations Are Associated with High CD49d Expression in Chronic Lymphocytic Leukemia
    Blood, 2014
    Co-Authors: Antonella Zucchetto, Riccardo Bomben, Francesca Rossi, Dania Benedetti, Chiara Caldana, Erika Tissino, Federico Pozzo, Silvia Rasi, Carmela Ciardullo, Francesco Zaja
    Abstract:

    Mutations of NOTCH1 have emerged as one of the most frequent somatic alterations in chronic lymphocytic leukemia (CLL), affecting up to 10–15% of patients. These mutations (~80% are 7544-7545delCT frameshift deletions) generate a truncated protein that accumulates in the cell and activates the downstream NOTCH1 signaling which is implicated in apoptosis resistance and increased survival of CLL cells. CD49d (α4 integrin chain) is one of the most relevant negative prognosticator in CLL, expressed by ~40% of CLL cases, and associated with aggressive/accelerated clinical courses, whose key role in CLL cell microenvironmental interactions has been thoroughly investigated. Literature data indicate that NOTCH1 has a role in activating the integrin signaling in several cell models. Given the higher CD49d expression characterizing trisomy 12 CLL, a CLL subset in which the NOTCH1 pathway is more often activated by NOTCH1 mutations, this study was aimed at investigating the contribution of NOTCH1 in the regulation of CD49d expression in CLL. The 7544-7545delCT NOTCH1 mutations were investigated by ARMS-PCR in 1027 CLL cases, all characterized for CD49d expression and for the cytogenetic profiles by FISH. NOTCH1 mutated cases were 158/1027 (15%), with a higher prevalence (36.7% of NOTCH1 mutated cases) in the trisomy 12 cytogenetic group. Analysis of CD49d expression highlighted a very strong association between the presence of NOTCH1 mutations and CD49d expression (p 30% of positive cells) was found in 102/158 (64.5%) NOTCH1 mutated cases as compared to 285/869 (32.8%) NOTCH1 wild-type cases. Of note, excluding trisomy 12 CLL, again NOTCH1 mutated CLL (100/865, 11.6%) displayed a significantly higher frequency of CD49d+ cases (52%) as compared to NOTCH1 wild-type CLL (25.7%) (p We next analyzed the percentage of mutated NOTCH1 DNA in the context of the CLL clone by a quantitative real-time PCR (QRT-PCR) approach set up by us to quantify the delCT NOTCH1 mutation. Using the 10% cut-off value to discriminate between cases with high (high NOTCH1) and low (low NOTCH1) mutation burden, 90/138 (67.7%) and 43/138 (32.3%) CLL cases were classified as low NOTCH1 and high NOTCH1, respectively. A higher prevalence of CD49d+ cases was found in the high NOTCH1 group (79%) as compared to the low NOTCH1 group (58%) (p=0.03). Moreover, a significant association between CD49d expression and a high NOTCH1 mutation burden was observed also excluding trisomy 12 CLL, with 69% of CD49d+ cases in the high NOTCH1 group, compared to 41% of CD49d+cases in the low NOTCH1 group (p=0.03). The association between NOTCH1 mutations and CD49d expression was next confirmed by next-generation sequencing results using the flow cytometrically sorted (>99% purity) CD49d- and CD49d+ components from 8 CLL cases characterized by both CD49d bimodal expression, and the presence of 7544-7545delCT NOTCH1 mutations at the subclonal level. In 7/8 cases, the CD49d+ component showed a higher NOTCH1 mutation burden compared to the CD49d- component, this difference reaching statistical significance in 4/7 cases. Of note, a similar clustering of mutations could not be observed in the CD49d- and CD49d+ components of other CLL cases characterized by bimodal CD49d expression and subclonal mutations of SF3B1 (n=1), BIRC3 (n=2) or TP53 (n=2). To verify whether NOTCH1 accumulation, as occurring in NOTCH1 mutated CLL, may influence CD49d expression, MEC-1 cells were transfected with a vector containing either a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or a NICD carrying a missense mutation (c.5304G>A) generating a stop codon at the beginning of the sequence, as control. The higher levels of both NOTCH1 transcript (fold increase over control=2.2) and protein (fold increase over control=1.3) characterizing mutated-NICD MEC-1 cells, was paralleled by higher levels of CD49d expression (mean fluorescence intensity=23.300 versus 12.400) in these cells. Altogether our data demonstrate a direct correlation between NOTCH1 mutations and CD49d expression also outside the trisomy 12 CLL group, and suggest that accumulation of NOTCH1 may be directly or indirectly responsible for the up-regulation of CD49d expression. Disclosures No relevant conflicts of interest to declare.

  • CD49d is overexpressed by trisomy 12 chronic lymphocytic leukemia cells: evidence for a methylation-dependent regulation mechanism.
    Blood, 2013
    Co-Authors: Antonella Zucchetto, Riccardo Bomben, Francesca Rossi, Dania Benedetti, Chiara Caldana, Erika Tissino, Michele Dal Bo, Evelyn Hutterer, Federico Pozzo, Giovanni D'arena
    Abstract:

    CD49d is a negative prognosticator in chronic lymphocytic leukemia (CLL), expressed by ~40% of CLL cases, and associated with aggressive and accelerated clinical courses. In the present study, analyzing CD49d expression in a wide CLL cohort (n=1200) belonging to different cytogenetic groups, we report that trisomy 12 CLL almost universally expressed CD49d, and were characterized by the highest CD49d expression levels among all CD49d + CLL. Through bisulfite genomic sequencing, we demonstrated that while CD49d + /trisomy12 CLL almost completely lacked methylation of the CD49d gene, CD49d - /no trisomy12 CLL were overall methylated, the methylation levels being inversely correlated to CD49d expression (p=0.0001). Consistently, CD49d expression was recovered in CD49d - hypermethylated CLL cells upon in-vitro treatment with the hypomethylating agent 5-aza-29-deoxycytidine. These findings may contribute to explain the clinico-biological features of trisomy 12 CLL, including the high rates of cell proliferation and disease progression, lymph node involvement and predisposition to Richter9s syndrome transformation.

  • CD49d Is Overexpressed in Trisomy 12 Chronic Lymphocytic Leukemia by an Epigenetic-Dependent Transcriptional Control
    Blood, 2012
    Co-Authors: Antonella Zucchetto, Riccardo Bomben, Francesca Rossi, Davide Rossi, Dania Benedetti, Chiara Caldana, Erika Tissino, Michele Dal Bo, Francesco Zaja, Gabriele Pozzato
    Abstract:

    Abstract 929 CD49d is an adhesion molecule with a variable expression in chronic lymphocytic leukemia (CLL). Only one third of CLL display CD49d expression in a significant fraction of cells, the remaining cases either expressing very low levels or completely lacking its expression. The negative prognostic impact of CD49d in CLL and its key role in microenvironmental interactions have been broadly described by several studies, whereas information on the mechanisms regulating CD49d expression in CLL is still lacking. Aiming to understand the genetic mechanisms underlying the different expression pattern of CD49d in CLL, CD49d expression was investigated by flow cytometry in the neoplastic component of 972 CLL patients, and correlated with cytogenetic profiles assessed by fluorescence in situ hybridization (FISH). Using the 30% positive cut-off value, 603 cases (62%) were CD49d-, whereas 369 cases (38%) were CD49d+. Correlation of CD49d expression with the major cytogenetic alterations (del13q14.3, trisomy 12, del11q22-q23-, del17p13.1), highlighted a very strong association between CD49d expression and the presence of trisomy 12 (p 99% purity) CD49d- and CD49d+ components from 3 CLL cases characterized by both CD49d bimodal expression (27%, 57%, and 75% of CD49d positive cells), and the presence of trisomy 12 in a fraction of the cells (25%, 42% and 63% respectively). In all cases, the CD49d- components almost completely lacked trisomy 12 (5%, 4%, and 0%), whereas the CD49d+ fractions displayed 70%, 62%, and 75% of trisomy 12, respectively (p A gene expression profiling was performed comparing CD49d+/trisomy12 (n=5) versus CD49d-/normal FISH CLL (n=5). According to bioinformatics tools for global analysis of gene function, the “Chromatin modification” (p Altogether, our results demonstrate that the overall down-regulated CD49d expression characterizing CLL is subjected to epigenetic control. Re-expression of CD49d may occur through a fine regulation of methyltransferase and chromatin modification processes, particularly active in trisomy 12 CLL, where CD49d expression may have a role in determining the clinical and biological features of this particular CLL subset. Disclosures: No relevant conflicts of interest to declare.

  • Low CD49d expression and long telomere identify a chronic lymphocytic leukemia subset with highly favourable outcome
    American journal of hematology, 2010
    Co-Authors: Davide Rossi, Antonella Zucchetto, Francesca Rossi, Valter Gattei, Silvia Rasi, Chiara Lobetti Bodoni, Lorenzo De Paoli, Marco Fangazio, Marco Ladetto, Gianluca Gaidano
    Abstract:

    CD49d expression and telomere length (TL) represent novel prognostic markers for chronic lymphocytic leukemia (CLL) [1―7]. The prognostic information carried by CD49d expression and TL in CLL has its rationale in the disease biology. CD49d is an adhesion molecule mediating cell-to-cell and cell-to-extracellular matrix interactions [8]. In CLL cells, CD49d transmits prosurvival/antlapoptotic signals from the tumor microenvironment to tumor cells [9]. Telomeres ensure genetic stability and regulate critical cellular functions, including proliferation and senescence [10]. In CLL, short TL associates with a fast proliferative history of the leukemic cells [1,11,12]. Both CD49d expression and short TL have been associated with increased genetic instability of the CLL clone [3,13]. From a clinical standpoint, a peculiar feature shared by CD49d expression and short TL is the association with CLL proliferation markers, including expression of CD38, high lactate dehydrogenase (LDH), high β-2-microglobulin, short time to lymphocyte doubling, and short time to progression to a more advanced stage [1―7]. Here, we tested whether the concomitant presence of high CD49d expression and short TL in the same CLL patient may help refine disease stratification for treatment prediction in patients that presented in early to intermediate Binet stage (Binet A and B) and that are candidate to watch and wait as initial management.

Riccardo Bomben - One of the best experts on this subject based on the ideXlab platform.

  • CD49d prevails over the novel recurrent mutations as independent prognosticator of overall survival in chronic lymphocytic leukemia
    Leukemia, 2016
    Co-Authors: M. Dal Bo, Antonella Zucchetto, D Benedetti, Riccardo Bomben, E Tissino, P Bulian, F M Rossi, F Pozzo, T Bittolo, P Nanni
    Abstract:

    CD49d, the alpha-chain of the integrin heterodimer α4β1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53 . In addition to TP53 , the clinical relevance of NOTCH1 , SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53 , NOTCH1 , SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P< 0.0001). Consistently, high CD49d expression identified CLL subsets with inferior OS in the context of each category of a previously reported hierarchical risk stratification model. Moreover, by evaluating the relative importance of biological prognosticators by random survival forests, CD49d was selected among the top-ranked OS predictor (variable importance =0.0410), along with IGHV mutational status and TP53 abnormalities. These results confirmed CD49d as an independent negative OS prognosticator in CLL also in comprehensive models comprising the novel recurrent mutations. In this context, TP53 disruption and NOTCH1 mutations retained prognostic relevance, in keeping with their roles in CLL cell immuno-chemoresistance.

  • Functional and clinical significance of the integrin alpha chain CD49d expression in chronic lymphocytic leukemia
    Current cancer drug targets, 2016
    Co-Authors: Michele Dal Bo, Riccardo Bomben, Francesca Rossi, Giovanni Del Poeta, Dania Benedetti, Chiara Caldana, Erika Tissino, Gianluca Gaidano, Pietro Bulian, Antonella Zucchetto
    Abstract:

    Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation/expansion of a clonal population of neoplastic cells with the morphological appearance of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. CD49d, the α chain of the α4β1 integrin heterodimer, is one of the main interactors between CLL cells and accessory cells in the microenvironmental sites and one of the main predictors of overall survival. In particular, CD49d is known to play a pivotal role in mediating both cell-cell and cell-matrix interactions in CLL-involved tissues eventually delivering prosurvival signals and protecting CLL cells from drug-induced damages. Treatment strategies targeting the α4β1 integrin could represent an interesting option in CLL. In this context, the recombinant anti-CD49d antibody natalizumab demonstrated the potential to overcome stromal cell-induced resistance of B cell lymphoma cells against cytotoxic drugs and rituximab in vitro. Moreover, a specific interest for the CD49d molecule raises from the clinical activity of the recently proposed inhibitors of kinases downstream the BCR that has been recently related with the inside-out activation of the α4β1 integrin. In the review, we addressed in detail the role of CD49d in CLL cells, including clinical impact, relationship with specific cytogenetic features, and CD49d-dependent interactions in lymph node and bone marrow microenvironment responsible for growth- and survival- supporting signals, eventually influencing CLL prognosis and therapeutic options.

  • NOTCH1 Mutations Are Associated with High CD49d Expression in Chronic Lymphocytic Leukemia
    Blood, 2014
    Co-Authors: Antonella Zucchetto, Riccardo Bomben, Francesca Rossi, Dania Benedetti, Chiara Caldana, Erika Tissino, Federico Pozzo, Silvia Rasi, Carmela Ciardullo, Francesco Zaja
    Abstract:

    Mutations of NOTCH1 have emerged as one of the most frequent somatic alterations in chronic lymphocytic leukemia (CLL), affecting up to 10–15% of patients. These mutations (~80% are 7544-7545delCT frameshift deletions) generate a truncated protein that accumulates in the cell and activates the downstream NOTCH1 signaling which is implicated in apoptosis resistance and increased survival of CLL cells. CD49d (α4 integrin chain) is one of the most relevant negative prognosticator in CLL, expressed by ~40% of CLL cases, and associated with aggressive/accelerated clinical courses, whose key role in CLL cell microenvironmental interactions has been thoroughly investigated. Literature data indicate that NOTCH1 has a role in activating the integrin signaling in several cell models. Given the higher CD49d expression characterizing trisomy 12 CLL, a CLL subset in which the NOTCH1 pathway is more often activated by NOTCH1 mutations, this study was aimed at investigating the contribution of NOTCH1 in the regulation of CD49d expression in CLL. The 7544-7545delCT NOTCH1 mutations were investigated by ARMS-PCR in 1027 CLL cases, all characterized for CD49d expression and for the cytogenetic profiles by FISH. NOTCH1 mutated cases were 158/1027 (15%), with a higher prevalence (36.7% of NOTCH1 mutated cases) in the trisomy 12 cytogenetic group. Analysis of CD49d expression highlighted a very strong association between the presence of NOTCH1 mutations and CD49d expression (p 30% of positive cells) was found in 102/158 (64.5%) NOTCH1 mutated cases as compared to 285/869 (32.8%) NOTCH1 wild-type cases. Of note, excluding trisomy 12 CLL, again NOTCH1 mutated CLL (100/865, 11.6%) displayed a significantly higher frequency of CD49d+ cases (52%) as compared to NOTCH1 wild-type CLL (25.7%) (p We next analyzed the percentage of mutated NOTCH1 DNA in the context of the CLL clone by a quantitative real-time PCR (QRT-PCR) approach set up by us to quantify the delCT NOTCH1 mutation. Using the 10% cut-off value to discriminate between cases with high (high NOTCH1) and low (low NOTCH1) mutation burden, 90/138 (67.7%) and 43/138 (32.3%) CLL cases were classified as low NOTCH1 and high NOTCH1, respectively. A higher prevalence of CD49d+ cases was found in the high NOTCH1 group (79%) as compared to the low NOTCH1 group (58%) (p=0.03). Moreover, a significant association between CD49d expression and a high NOTCH1 mutation burden was observed also excluding trisomy 12 CLL, with 69% of CD49d+ cases in the high NOTCH1 group, compared to 41% of CD49d+cases in the low NOTCH1 group (p=0.03). The association between NOTCH1 mutations and CD49d expression was next confirmed by next-generation sequencing results using the flow cytometrically sorted (>99% purity) CD49d- and CD49d+ components from 8 CLL cases characterized by both CD49d bimodal expression, and the presence of 7544-7545delCT NOTCH1 mutations at the subclonal level. In 7/8 cases, the CD49d+ component showed a higher NOTCH1 mutation burden compared to the CD49d- component, this difference reaching statistical significance in 4/7 cases. Of note, a similar clustering of mutations could not be observed in the CD49d- and CD49d+ components of other CLL cases characterized by bimodal CD49d expression and subclonal mutations of SF3B1 (n=1), BIRC3 (n=2) or TP53 (n=2). To verify whether NOTCH1 accumulation, as occurring in NOTCH1 mutated CLL, may influence CD49d expression, MEC-1 cells were transfected with a vector containing either a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or a NICD carrying a missense mutation (c.5304G>A) generating a stop codon at the beginning of the sequence, as control. The higher levels of both NOTCH1 transcript (fold increase over control=2.2) and protein (fold increase over control=1.3) characterizing mutated-NICD MEC-1 cells, was paralleled by higher levels of CD49d expression (mean fluorescence intensity=23.300 versus 12.400) in these cells. Altogether our data demonstrate a direct correlation between NOTCH1 mutations and CD49d expression also outside the trisomy 12 CLL group, and suggest that accumulation of NOTCH1 may be directly or indirectly responsible for the up-regulation of CD49d expression. Disclosures No relevant conflicts of interest to declare.

  • CD49d is overexpressed by trisomy 12 chronic lymphocytic leukemia cells: evidence for a methylation-dependent regulation mechanism.
    Blood, 2013
    Co-Authors: Antonella Zucchetto, Riccardo Bomben, Francesca Rossi, Dania Benedetti, Chiara Caldana, Erika Tissino, Michele Dal Bo, Evelyn Hutterer, Federico Pozzo, Giovanni D'arena
    Abstract:

    CD49d is a negative prognosticator in chronic lymphocytic leukemia (CLL), expressed by ~40% of CLL cases, and associated with aggressive and accelerated clinical courses. In the present study, analyzing CD49d expression in a wide CLL cohort (n=1200) belonging to different cytogenetic groups, we report that trisomy 12 CLL almost universally expressed CD49d, and were characterized by the highest CD49d expression levels among all CD49d + CLL. Through bisulfite genomic sequencing, we demonstrated that while CD49d + /trisomy12 CLL almost completely lacked methylation of the CD49d gene, CD49d - /no trisomy12 CLL were overall methylated, the methylation levels being inversely correlated to CD49d expression (p=0.0001). Consistently, CD49d expression was recovered in CD49d - hypermethylated CLL cells upon in-vitro treatment with the hypomethylating agent 5-aza-29-deoxycytidine. These findings may contribute to explain the clinico-biological features of trisomy 12 CLL, including the high rates of cell proliferation and disease progression, lymph node involvement and predisposition to Richter9s syndrome transformation.

  • Circulating CLL Cells Expressing CD49d Display a Phospho-Proteomic Profile Consistent with a Constitutive Receptor Engagement by Blood-Borne Ligands
    Blood, 2012
    Co-Authors: Dania Benedetti, Riccardo Bomben, Daniela Marconi, Davide Rossi, Silvia Deaglio, Erika Tissino, Michele Dal Bo, Tiziana Vaisitti, Gianluca Gaidano, Gabriele Pozzato
    Abstract:

    Abstract 930 Chronic lymphocytic leukemia (CLL) cell growth and survival occur in defined microenvironment niches controlled by several receptor/ligand interactions including those mediated by the α4β1 integrin. This integrin, in particular, is known to interact with both the extracellular matrix component fibronectin, via the non-RGD CS-1 fragments, and the bone marrow/stromal components, via the vascular-cell adhesion molecule-1 (VCAM-1). The α4 integrin chain (a.k.a. CD49d) has been previously shown to be associated with poor prognosis in CLL by marking a subset of CLL patients characterized by a more aggressive clinical course both in term of disease progression and overall survival. Despite the great deal of studies investigating CLL cell microenvironmental interactions in tissue sites, little is known regarding the constitutive engagement of adhesion receptors in circulating CLL cells, and the role in this context of plasma/plasma components. To address this issue, the proteomic profiles of circulating leukemic cells from 80 CLL cases expressing or not CD49d were explored using a reverse phase protein microarray (RPMA) approach quantitatively analysing 77 proteins (61 phosphoproteins) known to be involved in translational control, cell growth, apoptosis, B cell receptor and cytoskeletal signaling. Comparison of the signaling activation portrait between 40 CD49d pos and 40 CD49d neg CLL cases highlighted the over-expression in CD49d pos CLL of proteins involved in the regulation of integrin-mediated cytoskeletal dynamics, such as phospho-p21-activated kinase (PAK1 Ser199-204/PAK2 Ser192-197; p=0.0005), phospho-LIM kinase (LIMK1 Thr508/LIMK2 Thr505; p=0.00001) and the adaptor protein CrkII Tyr221 (p=0.039). Moreover, CD49d pos CLL cells were characterized by a high correlation between proteins involved in integrin-mediated signal trasduction, including the focal-adhesion kinase (FAK Tyr576-Ser577), the tyrosine kinase Src Tyr527 and the adaptor protein CrkL Tyr207. Since PAK and LIMK represent key players in the modulation of the structure and the activity of actin cytoskeleton, we focused on these proteins for validation experiments. The constitutive pPAK and pLIMK overexpression in the CD49d pos CLL group was confirmed by western blot analysis comparing purified CLL cells from 3 CD49d pos versus 3 CD49d neg cases (mean fold increase >100 for both proteins). The results obtained suggest that integrin signalling is constitutively active in CD49d-expressing circulating CLL cells, pointing to a constitutive receptor engagement by CD49d ligands allegedly present in plasma. To test whether plasma constituents could modulate integrin-signaling proteins, CLL cells from 5 cases, expressing or not CD49d, were challenged with autologous plasma (1:3 dilution) for 30 seconds, 1 and 3 minutes. The presence of plasma induced a strong increase of PAK and LIMK phosphorylation intensities in CD49d pos CLL cells, starting at 30 seconds upon stimulation (mean fold increase >10 as compared to control for both proteins), and increasing after 1 and 3 minutes (mean fold increase >40 and >50 for both the proteins, respectively). Conversely, plasma stimulation did not induce pPAK and pLIMK expression modulation in CD49d neg CLL cells. Of note, pretreatment of CD49d-expressing CLL cells with the anti-CD49d HP1/2 blocking antibody, resulted in lower up-regulation of pPAK and pLIMK with an overall 60% inhibition for both the proteins (p=0.01), confirming the involvement of CD49d triggering in the observed activated signaling. Given the above results, we investigated plasma from 24 CLL patients, expressing CD49d at different levels, for the presence of the CD49d ligand fibronectin using an ELISA detection kit. All samples tested showed more than 1 mg/ml fibronectin concentration, without differences between CD49d-expressing and CD49d neg CLL cases. Altogether these results sustain the hypothesis of an active role of plasma/plasma components in the activation of CD49d-mediated integrin pathway, thus favoring the delivery of chemoresistance/pro-survival signals even in the context of circulating CLL cells. Our results may be of interest in the perspective of novel therapies (e.g. Bruton tyrosine kinase inhibitors) known to provoke a massive egress of neoplastic cells from tissue sites into the blood stream. Disclosures: No relevant conflicts of interest to declare.

Davide Rossi - One of the best experts on this subject based on the ideXlab platform.

  • CD49d Is Overexpressed in Trisomy 12 Chronic Lymphocytic Leukemia by an Epigenetic-Dependent Transcriptional Control
    Blood, 2012
    Co-Authors: Antonella Zucchetto, Riccardo Bomben, Francesca Rossi, Davide Rossi, Dania Benedetti, Chiara Caldana, Erika Tissino, Michele Dal Bo, Francesco Zaja, Gabriele Pozzato
    Abstract:

    Abstract 929 CD49d is an adhesion molecule with a variable expression in chronic lymphocytic leukemia (CLL). Only one third of CLL display CD49d expression in a significant fraction of cells, the remaining cases either expressing very low levels or completely lacking its expression. The negative prognostic impact of CD49d in CLL and its key role in microenvironmental interactions have been broadly described by several studies, whereas information on the mechanisms regulating CD49d expression in CLL is still lacking. Aiming to understand the genetic mechanisms underlying the different expression pattern of CD49d in CLL, CD49d expression was investigated by flow cytometry in the neoplastic component of 972 CLL patients, and correlated with cytogenetic profiles assessed by fluorescence in situ hybridization (FISH). Using the 30% positive cut-off value, 603 cases (62%) were CD49d-, whereas 369 cases (38%) were CD49d+. Correlation of CD49d expression with the major cytogenetic alterations (del13q14.3, trisomy 12, del11q22-q23-, del17p13.1), highlighted a very strong association between CD49d expression and the presence of trisomy 12 (p 99% purity) CD49d- and CD49d+ components from 3 CLL cases characterized by both CD49d bimodal expression (27%, 57%, and 75% of CD49d positive cells), and the presence of trisomy 12 in a fraction of the cells (25%, 42% and 63% respectively). In all cases, the CD49d- components almost completely lacked trisomy 12 (5%, 4%, and 0%), whereas the CD49d+ fractions displayed 70%, 62%, and 75% of trisomy 12, respectively (p A gene expression profiling was performed comparing CD49d+/trisomy12 (n=5) versus CD49d-/normal FISH CLL (n=5). According to bioinformatics tools for global analysis of gene function, the “Chromatin modification” (p Altogether, our results demonstrate that the overall down-regulated CD49d expression characterizing CLL is subjected to epigenetic control. Re-expression of CD49d may occur through a fine regulation of methyltransferase and chromatin modification processes, particularly active in trisomy 12 CLL, where CD49d expression may have a role in determining the clinical and biological features of this particular CLL subset. Disclosures: No relevant conflicts of interest to declare.

  • Circulating CLL Cells Expressing CD49d Display a Phospho-Proteomic Profile Consistent with a Constitutive Receptor Engagement by Blood-Borne Ligands
    Blood, 2012
    Co-Authors: Dania Benedetti, Riccardo Bomben, Daniela Marconi, Davide Rossi, Silvia Deaglio, Erika Tissino, Michele Dal Bo, Tiziana Vaisitti, Gianluca Gaidano, Gabriele Pozzato
    Abstract:

    Abstract 930 Chronic lymphocytic leukemia (CLL) cell growth and survival occur in defined microenvironment niches controlled by several receptor/ligand interactions including those mediated by the α4β1 integrin. This integrin, in particular, is known to interact with both the extracellular matrix component fibronectin, via the non-RGD CS-1 fragments, and the bone marrow/stromal components, via the vascular-cell adhesion molecule-1 (VCAM-1). The α4 integrin chain (a.k.a. CD49d) has been previously shown to be associated with poor prognosis in CLL by marking a subset of CLL patients characterized by a more aggressive clinical course both in term of disease progression and overall survival. Despite the great deal of studies investigating CLL cell microenvironmental interactions in tissue sites, little is known regarding the constitutive engagement of adhesion receptors in circulating CLL cells, and the role in this context of plasma/plasma components. To address this issue, the proteomic profiles of circulating leukemic cells from 80 CLL cases expressing or not CD49d were explored using a reverse phase protein microarray (RPMA) approach quantitatively analysing 77 proteins (61 phosphoproteins) known to be involved in translational control, cell growth, apoptosis, B cell receptor and cytoskeletal signaling. Comparison of the signaling activation portrait between 40 CD49d pos and 40 CD49d neg CLL cases highlighted the over-expression in CD49d pos CLL of proteins involved in the regulation of integrin-mediated cytoskeletal dynamics, such as phospho-p21-activated kinase (PAK1 Ser199-204/PAK2 Ser192-197; p=0.0005), phospho-LIM kinase (LIMK1 Thr508/LIMK2 Thr505; p=0.00001) and the adaptor protein CrkII Tyr221 (p=0.039). Moreover, CD49d pos CLL cells were characterized by a high correlation between proteins involved in integrin-mediated signal trasduction, including the focal-adhesion kinase (FAK Tyr576-Ser577), the tyrosine kinase Src Tyr527 and the adaptor protein CrkL Tyr207. Since PAK and LIMK represent key players in the modulation of the structure and the activity of actin cytoskeleton, we focused on these proteins for validation experiments. The constitutive pPAK and pLIMK overexpression in the CD49d pos CLL group was confirmed by western blot analysis comparing purified CLL cells from 3 CD49d pos versus 3 CD49d neg cases (mean fold increase >100 for both proteins). The results obtained suggest that integrin signalling is constitutively active in CD49d-expressing circulating CLL cells, pointing to a constitutive receptor engagement by CD49d ligands allegedly present in plasma. To test whether plasma constituents could modulate integrin-signaling proteins, CLL cells from 5 cases, expressing or not CD49d, were challenged with autologous plasma (1:3 dilution) for 30 seconds, 1 and 3 minutes. The presence of plasma induced a strong increase of PAK and LIMK phosphorylation intensities in CD49d pos CLL cells, starting at 30 seconds upon stimulation (mean fold increase >10 as compared to control for both proteins), and increasing after 1 and 3 minutes (mean fold increase >40 and >50 for both the proteins, respectively). Conversely, plasma stimulation did not induce pPAK and pLIMK expression modulation in CD49d neg CLL cells. Of note, pretreatment of CD49d-expressing CLL cells with the anti-CD49d HP1/2 blocking antibody, resulted in lower up-regulation of pPAK and pLIMK with an overall 60% inhibition for both the proteins (p=0.01), confirming the involvement of CD49d triggering in the observed activated signaling. Given the above results, we investigated plasma from 24 CLL patients, expressing CD49d at different levels, for the presence of the CD49d ligand fibronectin using an ELISA detection kit. All samples tested showed more than 1 mg/ml fibronectin concentration, without differences between CD49d-expressing and CD49d neg CLL cases. Altogether these results sustain the hypothesis of an active role of plasma/plasma components in the activation of CD49d-mediated integrin pathway, thus favoring the delivery of chemoresistance/pro-survival signals even in the context of circulating CLL cells. Our results may be of interest in the perspective of novel therapies (e.g. Bruton tyrosine kinase inhibitors) known to provoke a massive egress of neoplastic cells from tissue sites into the blood stream. Disclosures: No relevant conflicts of interest to declare.

  • Prognostic Relevance of CD49d Expression On B Leukemic Cells in Chronic Lymphocytic Leukemia. Meta-Analysis of Published and unpublished Individual Data From 3146 Patients
    Blood, 2012
    Co-Authors: Pietro Bulian, Davide Rossi, Giovanni Del Poeta, Tait D. Shanafelt, Gianluca Gaidano, Chris Pepper, Christopher Fegan, Luca Baldini, L. Cro, Holger Nückel
    Abstract:

    Abstract 3871 Background. A number of investigators have provided data supporting the value of CD49d expression on B leukemic cells in chronic lymphocytic leukemia (CLL) as independent prognostic variable. These studies used a variety of clinical end points including overall survival (OS), time to treatment (TTT) or treatment free survival (TFS). The factors included in multivariate analyses of these studies also differed. Unresolved issues regarding the prognostic value of CD49d assessment in CLL included the choice of the optimal cut-off to define positivity and CD49d prognostic value in patients subsets defined by other standard prognostic factors. Aim. To perform meta-analysis using individual patient level data from studies of CD49d expression to evaluate its ability to predict OS (primary end-point) and TTT/TFS (secondary end-point). Methods. Studies published by 30 April 2011, reporting an association of high CD49d expression on B CLL cells, measured by flow cytometry, and end-points were identified by Medline search. Additionally, we performed a manual review of abstracts presented at the congress of the American Society of Hematology from 2006 to 2010. CD49d was used as a categorical variable, as coded in original studies. Results. We identified 6 published studies and one abstract for inclusion. All authors agreed to provide individual data on the patients in these publications as well as unpublished data on additional patients to be used for replication analysis. Four authors also submitted updated follow-up data. We collected the following variables: date of diagnosis, OS, TTT/TFS, CD49d, CD38, ZAP-70, immunoglobulin mutational (IGHV) status, del17p and del11q chromosomal aberrations, age, stage, ALC, and β2 microglobulin concentration. Data from 3146 individual patients was available with 261 subsequently excluded due to missing end point and/or CD49d data. Of 2771 patients with valid data, 1405 (51%) were included in the previous publications and 1366 (49%) were unpublished. Before starting analyses a decision was made to perform meta-analysis on published data and use data from previously unpublished data as a validation cohort. Pooled CD49d hazard ratio for OS was 2.62 (1.88–3.64). In bivariate analysis, the prognostic value of CD49d was confirmed and of comparable value in patients subsets defined by CD38 and ZAP-70 expression, IGHV status, unfavorable chromosomal aberrations. Finally, we performed a multivariate analysis including CD49d, CD38, ZAP-70, IGHV status, del17p and del11q. CD49d was significantly associated with shorter overall survival in this adjusted model, with a hazard ratio of 2.47(1.17–5.21). Inspection of Martingale residuals plots of CD49d in each study failed to show a recognizable cut-point. CD49d cut-points previously adopted in published studies (30% and 45%) were subjected to validation in the cohort of 1366 unpublished patients. The Cox model with 45% cut-off had a slight greater hazard ratio (2.73 vs 2.65, log-rank p1400 patients confirm the association of high CD49d expression with short OS independent of other prognostic parameters. While the 45% cut-off has a marginal better prognostic power, the 30% cut-off has the advantage of being already in use for CD38 and generally adopted for other surface antigens in chronic leukemia immunophenotyping. These findings may have implication for patient's stratification in future prospective studies and potential therapeutic efforts to target CD49d or CD49d signaling. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Burger: Pharmacyclics: Consultancy, Research Funding.

  • prognostic relevance of CD49d expression on b leukemic cells in chronic lymphocytic leukemia meta analysis of published and unpublished individual data from 3146 patients
    Blood, 2012
    Co-Authors: Pietro Bulian, Davide Rossi, Giovanni Del Poeta, Tait D. Shanafelt, Gianluca Gaidano, Chris Pepper, Christopher Fegan, Luca Baldini, L. Cro, Holger Nückel
    Abstract:

    Abstract 3871 Background. A number of investigators have provided data supporting the value of CD49d expression on B leukemic cells in chronic lymphocytic leukemia (CLL) as independent prognostic variable. These studies used a variety of clinical end points including overall survival (OS), time to treatment (TTT) or treatment free survival (TFS). The factors included in multivariate analyses of these studies also differed. Unresolved issues regarding the prognostic value of CD49d assessment in CLL included the choice of the optimal cut-off to define positivity and CD49d prognostic value in patients subsets defined by other standard prognostic factors. Aim. To perform meta-analysis using individual patient level data from studies of CD49d expression to evaluate its ability to predict OS (primary end-point) and TTT/TFS (secondary end-point). Methods. Studies published by 30 April 2011, reporting an association of high CD49d expression on B CLL cells, measured by flow cytometry, and end-points were identified by Medline search. Additionally, we performed a manual review of abstracts presented at the congress of the American Society of Hematology from 2006 to 2010. CD49d was used as a categorical variable, as coded in original studies. Results. We identified 6 published studies and one abstract for inclusion. All authors agreed to provide individual data on the patients in these publications as well as unpublished data on additional patients to be used for replication analysis. Four authors also submitted updated follow-up data. We collected the following variables: date of diagnosis, OS, TTT/TFS, CD49d, CD38, ZAP-70, immunoglobulin mutational (IGHV) status, del17p and del11q chromosomal aberrations, age, stage, ALC, and β2 microglobulin concentration. Data from 3146 individual patients was available with 261 subsequently excluded due to missing end point and/or CD49d data. Of 2771 patients with valid data, 1405 (51%) were included in the previous publications and 1366 (49%) were unpublished. Before starting analyses a decision was made to perform meta-analysis on published data and use data from previously unpublished data as a validation cohort. Pooled CD49d hazard ratio for OS was 2.62 (1.88–3.64). In bivariate analysis, the prognostic value of CD49d was confirmed and of comparable value in patients subsets defined by CD38 and ZAP-70 expression, IGHV status, unfavorable chromosomal aberrations. Finally, we performed a multivariate analysis including CD49d, CD38, ZAP-70, IGHV status, del17p and del11q. CD49d was significantly associated with shorter overall survival in this adjusted model, with a hazard ratio of 2.47(1.17–5.21). Inspection of Martingale residuals plots of CD49d in each study failed to show a recognizable cut-point. CD49d cut-points previously adopted in published studies (30% and 45%) were subjected to validation in the cohort of 1366 unpublished patients. The Cox model with 45% cut-off had a slight greater hazard ratio (2.73 vs 2.65, log-rank p<0.0001). Only 4% of patients changed prognostic category after swapping the cut-off from 45% to 30%. Conclusions. Preliminary results of a meta-analysis using individual patient data from >1400 patients confirm the association of high CD49d expression with short OS independent of other prognostic parameters. While the 45% cut-off has a marginal better prognostic power, the 30% cut-off has the advantage of being already in use for CD38 and generally adopted for other surface antigens in chronic leukemia immunophenotyping. These findings may have implication for patient's stratification in future prospective studies and potential therapeutic efforts to target CD49d or CD49d signaling. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Burger: Pharmacyclics: Consultancy, Research Funding.

  • The Elastin Microfibril Interfacer-1 (EMILIN-1) Is a Ligand for CD49d in Chronic Lymphocytic Leukemia Cells
    Blood, 2012
    Co-Authors: Erika Tissino, Riccardo Bomben, Davide Rossi, Silvia Deaglio, Dania Benedetti, Michele Dal Bo, Francesco Zaja, Gabriele Pozzato, Tiziana Vaisitti, Francesco Di Raimondo
    Abstract:

    Abstract 1772 CD49d (α4 integrin chain) is a strong negative prognosticator in chronic lymphocytic leukemia (CLL) with a key role in CLL cell microenvironmental interactions. CD49d triggering by its main ligands Vascular Cell Adhesion Molecule-1 (VCAM-1) and non-RGD sites (CS-1 fragments) of fibronectin (FN) activates signalling pathways delivering pro-survival signals, and promoting resistance to drug-induced apoptosis in CLL. Recently, the globular (g) C1q-like domain of Elastin MIcrofibriL INterfacer1 (EMILIN1), an adhesive extracellular matrix constituent, was described as a new ligand for CD49d, where it operates as a negative modulator of proliferation signals in substrate-adherent non-hematopoietic CD49d+ cells (Danussi et al, J Cell Biol, 2011). Here, we investigated the distribution of EMILIN1 in normal and CLL-involved tissues, and the effects of EMILIN1/CD49d interaction in CLL in terms of adhesion and survival. By taking advantage of a specific anti-human EMILIN1 monoclonal antibody (Spessotto et al, J Biol Chem, 2003), exploratory staining in reactive lymphoid tissues (tonsil) indicated a clear extracellular EMILIN1 specific reactivity in the outer zone of the mantle/marginal areas. When investigated in lymph node tissues from CLL cases (n=3) by both immunohistochemical and immunofluorescence analyses, a clear EMILIN1 positive staining was detected intermingled with the neoplastic component. To verify whether EMILIN1 could promote CLL cell adhesion, we took advantage of the CLL-derived CD49d+ Mec-1 CLL-like cell model, previously demonstrated by us to adhere to both VCAM-1 and FN (Zucchetto et al, Leukemia, 2012). Results demonstrated similar adhesion levels of Mec-1 cells on VCAM-1, CS-1 fragment of FN and the gC1q-like EMILIN1 domain (mean number of adherent cells per field 267±24, 272±7 and 317±21, respectively).Consistently, adhesion experiments with primary CLL cells characterized by high and homogeneous CD49d expression confirmed similar levels of adhesion on both VCAM-1 and EMILIN1 (mean number of adherent cells per field 141±55 and 135±76, respectively). In all cases adhesion was specifically blocked by pre-treatment with the anti-CD49d HP1/2 blocking antibody. Immunofluorescence analysis with Mec-1 and primary CLL cells showed the recruitment of phospho-Vav-1 at the CD49d/EMILIN1 adhesion sites, with concomitant F-actin reorganization, confirming the activation of the integrin signalling pathway. The effects of EMILIN1/CD49d interactions in CLL were next investigated performing short-term adhesion experiments (2 and 5 minutes) on gC1q-like EMILIN1 domain and VCAM-1 using CLL cells from three CD49d+ cases, and verifying the phosphorylation of Akt and ERK1/2 kinases, key mediators of survival signals by western blotting. In all cases, an increased intensity of Akt and ERK1/2 phosphorylation was documented after two-minutes of adhesion on both EMILIN1 (mean fold increase =1.2±0.1, and =5.0±1.0 as compared to controls, respectively) and VCAM-1 (mean fold increase =1.6±0.2, and =4.0±1.9, respectively). The phosphorylation was even greater after five minutes (mean fold increase =2.4±0.5, and =10.0±4.0, for EMILIN1 and =2.4±0.6 and =5.0±0.7 for VCAM-1, respectively). The concomitant increase of phospho-Vav-1 confirmed the activation of the integrin signalling pathway. Finally, we verified whether CD49d/EMILIN1 interaction was able to protect CLL cells from spontaneous apoptosis, by culturing purified cells from 6 CLL cases on gC1q-like EMILIN1 domain, VCAM-1, or control substrate (1% BSA), and checking cell viability after 5 days. Both VCAM-1 and EMILIN1 were able to protect CLL cells from spontaneous apoptosis (p=0.03 and p=0.001, respectively), the viability obtained on EMILIN1 being also significantly higher than that observed on VCAM-1 (p=0.007). In conclusion, we showed here for the first time that EMILIN1 is present in normal and CLL-involved lymphoid tissues, and it is able to efficiently bind to CD49d, as expressed by CLL cells. At variance of what demonstrated in non-hematopoietic models, EMILIN1 was shown to be able to deliver anti-apoptotic/pro-survival signals to circulating CLL cells. EMILIN1/CD49d interactions may have a role in the maintenance of the neoplastic clone in CD49d-expressing CLL. Disclosures: No relevant conflicts of interest to declare.

Dania Benedetti - One of the best experts on this subject based on the ideXlab platform.

  • Functional and clinical significance of the integrin alpha chain CD49d expression in chronic lymphocytic leukemia
    Current cancer drug targets, 2016
    Co-Authors: Michele Dal Bo, Riccardo Bomben, Francesca Rossi, Giovanni Del Poeta, Dania Benedetti, Chiara Caldana, Erika Tissino, Gianluca Gaidano, Pietro Bulian, Antonella Zucchetto
    Abstract:

    Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation/expansion of a clonal population of neoplastic cells with the morphological appearance of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. CD49d, the α chain of the α4β1 integrin heterodimer, is one of the main interactors between CLL cells and accessory cells in the microenvironmental sites and one of the main predictors of overall survival. In particular, CD49d is known to play a pivotal role in mediating both cell-cell and cell-matrix interactions in CLL-involved tissues eventually delivering prosurvival signals and protecting CLL cells from drug-induced damages. Treatment strategies targeting the α4β1 integrin could represent an interesting option in CLL. In this context, the recombinant anti-CD49d antibody natalizumab demonstrated the potential to overcome stromal cell-induced resistance of B cell lymphoma cells against cytotoxic drugs and rituximab in vitro. Moreover, a specific interest for the CD49d molecule raises from the clinical activity of the recently proposed inhibitors of kinases downstream the BCR that has been recently related with the inside-out activation of the α4β1 integrin. In the review, we addressed in detail the role of CD49d in CLL cells, including clinical impact, relationship with specific cytogenetic features, and CD49d-dependent interactions in lymph node and bone marrow microenvironment responsible for growth- and survival- supporting signals, eventually influencing CLL prognosis and therapeutic options.

  • NOTCH1 Mutations Are Associated with High CD49d Expression in Chronic Lymphocytic Leukemia
    Blood, 2014
    Co-Authors: Antonella Zucchetto, Riccardo Bomben, Francesca Rossi, Dania Benedetti, Chiara Caldana, Erika Tissino, Federico Pozzo, Silvia Rasi, Carmela Ciardullo, Francesco Zaja
    Abstract:

    Mutations of NOTCH1 have emerged as one of the most frequent somatic alterations in chronic lymphocytic leukemia (CLL), affecting up to 10–15% of patients. These mutations (~80% are 7544-7545delCT frameshift deletions) generate a truncated protein that accumulates in the cell and activates the downstream NOTCH1 signaling which is implicated in apoptosis resistance and increased survival of CLL cells. CD49d (α4 integrin chain) is one of the most relevant negative prognosticator in CLL, expressed by ~40% of CLL cases, and associated with aggressive/accelerated clinical courses, whose key role in CLL cell microenvironmental interactions has been thoroughly investigated. Literature data indicate that NOTCH1 has a role in activating the integrin signaling in several cell models. Given the higher CD49d expression characterizing trisomy 12 CLL, a CLL subset in which the NOTCH1 pathway is more often activated by NOTCH1 mutations, this study was aimed at investigating the contribution of NOTCH1 in the regulation of CD49d expression in CLL. The 7544-7545delCT NOTCH1 mutations were investigated by ARMS-PCR in 1027 CLL cases, all characterized for CD49d expression and for the cytogenetic profiles by FISH. NOTCH1 mutated cases were 158/1027 (15%), with a higher prevalence (36.7% of NOTCH1 mutated cases) in the trisomy 12 cytogenetic group. Analysis of CD49d expression highlighted a very strong association between the presence of NOTCH1 mutations and CD49d expression (p 30% of positive cells) was found in 102/158 (64.5%) NOTCH1 mutated cases as compared to 285/869 (32.8%) NOTCH1 wild-type cases. Of note, excluding trisomy 12 CLL, again NOTCH1 mutated CLL (100/865, 11.6%) displayed a significantly higher frequency of CD49d+ cases (52%) as compared to NOTCH1 wild-type CLL (25.7%) (p We next analyzed the percentage of mutated NOTCH1 DNA in the context of the CLL clone by a quantitative real-time PCR (QRT-PCR) approach set up by us to quantify the delCT NOTCH1 mutation. Using the 10% cut-off value to discriminate between cases with high (high NOTCH1) and low (low NOTCH1) mutation burden, 90/138 (67.7%) and 43/138 (32.3%) CLL cases were classified as low NOTCH1 and high NOTCH1, respectively. A higher prevalence of CD49d+ cases was found in the high NOTCH1 group (79%) as compared to the low NOTCH1 group (58%) (p=0.03). Moreover, a significant association between CD49d expression and a high NOTCH1 mutation burden was observed also excluding trisomy 12 CLL, with 69% of CD49d+ cases in the high NOTCH1 group, compared to 41% of CD49d+cases in the low NOTCH1 group (p=0.03). The association between NOTCH1 mutations and CD49d expression was next confirmed by next-generation sequencing results using the flow cytometrically sorted (>99% purity) CD49d- and CD49d+ components from 8 CLL cases characterized by both CD49d bimodal expression, and the presence of 7544-7545delCT NOTCH1 mutations at the subclonal level. In 7/8 cases, the CD49d+ component showed a higher NOTCH1 mutation burden compared to the CD49d- component, this difference reaching statistical significance in 4/7 cases. Of note, a similar clustering of mutations could not be observed in the CD49d- and CD49d+ components of other CLL cases characterized by bimodal CD49d expression and subclonal mutations of SF3B1 (n=1), BIRC3 (n=2) or TP53 (n=2). To verify whether NOTCH1 accumulation, as occurring in NOTCH1 mutated CLL, may influence CD49d expression, MEC-1 cells were transfected with a vector containing either a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or a NICD carrying a missense mutation (c.5304G>A) generating a stop codon at the beginning of the sequence, as control. The higher levels of both NOTCH1 transcript (fold increase over control=2.2) and protein (fold increase over control=1.3) characterizing mutated-NICD MEC-1 cells, was paralleled by higher levels of CD49d expression (mean fluorescence intensity=23.300 versus 12.400) in these cells. Altogether our data demonstrate a direct correlation between NOTCH1 mutations and CD49d expression also outside the trisomy 12 CLL group, and suggest that accumulation of NOTCH1 may be directly or indirectly responsible for the up-regulation of CD49d expression. Disclosures No relevant conflicts of interest to declare.

  • CD49d is overexpressed by trisomy 12 chronic lymphocytic leukemia cells: evidence for a methylation-dependent regulation mechanism.
    Blood, 2013
    Co-Authors: Antonella Zucchetto, Riccardo Bomben, Francesca Rossi, Dania Benedetti, Chiara Caldana, Erika Tissino, Michele Dal Bo, Evelyn Hutterer, Federico Pozzo, Giovanni D'arena
    Abstract:

    CD49d is a negative prognosticator in chronic lymphocytic leukemia (CLL), expressed by ~40% of CLL cases, and associated with aggressive and accelerated clinical courses. In the present study, analyzing CD49d expression in a wide CLL cohort (n=1200) belonging to different cytogenetic groups, we report that trisomy 12 CLL almost universally expressed CD49d, and were characterized by the highest CD49d expression levels among all CD49d + CLL. Through bisulfite genomic sequencing, we demonstrated that while CD49d + /trisomy12 CLL almost completely lacked methylation of the CD49d gene, CD49d - /no trisomy12 CLL were overall methylated, the methylation levels being inversely correlated to CD49d expression (p=0.0001). Consistently, CD49d expression was recovered in CD49d - hypermethylated CLL cells upon in-vitro treatment with the hypomethylating agent 5-aza-29-deoxycytidine. These findings may contribute to explain the clinico-biological features of trisomy 12 CLL, including the high rates of cell proliferation and disease progression, lymph node involvement and predisposition to Richter9s syndrome transformation.

  • CD49d Is Overexpressed in Trisomy 12 Chronic Lymphocytic Leukemia by an Epigenetic-Dependent Transcriptional Control
    Blood, 2012
    Co-Authors: Antonella Zucchetto, Riccardo Bomben, Francesca Rossi, Davide Rossi, Dania Benedetti, Chiara Caldana, Erika Tissino, Michele Dal Bo, Francesco Zaja, Gabriele Pozzato
    Abstract:

    Abstract 929 CD49d is an adhesion molecule with a variable expression in chronic lymphocytic leukemia (CLL). Only one third of CLL display CD49d expression in a significant fraction of cells, the remaining cases either expressing very low levels or completely lacking its expression. The negative prognostic impact of CD49d in CLL and its key role in microenvironmental interactions have been broadly described by several studies, whereas information on the mechanisms regulating CD49d expression in CLL is still lacking. Aiming to understand the genetic mechanisms underlying the different expression pattern of CD49d in CLL, CD49d expression was investigated by flow cytometry in the neoplastic component of 972 CLL patients, and correlated with cytogenetic profiles assessed by fluorescence in situ hybridization (FISH). Using the 30% positive cut-off value, 603 cases (62%) were CD49d-, whereas 369 cases (38%) were CD49d+. Correlation of CD49d expression with the major cytogenetic alterations (del13q14.3, trisomy 12, del11q22-q23-, del17p13.1), highlighted a very strong association between CD49d expression and the presence of trisomy 12 (p 99% purity) CD49d- and CD49d+ components from 3 CLL cases characterized by both CD49d bimodal expression (27%, 57%, and 75% of CD49d positive cells), and the presence of trisomy 12 in a fraction of the cells (25%, 42% and 63% respectively). In all cases, the CD49d- components almost completely lacked trisomy 12 (5%, 4%, and 0%), whereas the CD49d+ fractions displayed 70%, 62%, and 75% of trisomy 12, respectively (p A gene expression profiling was performed comparing CD49d+/trisomy12 (n=5) versus CD49d-/normal FISH CLL (n=5). According to bioinformatics tools for global analysis of gene function, the “Chromatin modification” (p Altogether, our results demonstrate that the overall down-regulated CD49d expression characterizing CLL is subjected to epigenetic control. Re-expression of CD49d may occur through a fine regulation of methyltransferase and chromatin modification processes, particularly active in trisomy 12 CLL, where CD49d expression may have a role in determining the clinical and biological features of this particular CLL subset. Disclosures: No relevant conflicts of interest to declare.

  • Circulating CLL Cells Expressing CD49d Display a Phospho-Proteomic Profile Consistent with a Constitutive Receptor Engagement by Blood-Borne Ligands
    Blood, 2012
    Co-Authors: Dania Benedetti, Riccardo Bomben, Daniela Marconi, Davide Rossi, Silvia Deaglio, Erika Tissino, Michele Dal Bo, Tiziana Vaisitti, Gianluca Gaidano, Gabriele Pozzato
    Abstract:

    Abstract 930 Chronic lymphocytic leukemia (CLL) cell growth and survival occur in defined microenvironment niches controlled by several receptor/ligand interactions including those mediated by the α4β1 integrin. This integrin, in particular, is known to interact with both the extracellular matrix component fibronectin, via the non-RGD CS-1 fragments, and the bone marrow/stromal components, via the vascular-cell adhesion molecule-1 (VCAM-1). The α4 integrin chain (a.k.a. CD49d) has been previously shown to be associated with poor prognosis in CLL by marking a subset of CLL patients characterized by a more aggressive clinical course both in term of disease progression and overall survival. Despite the great deal of studies investigating CLL cell microenvironmental interactions in tissue sites, little is known regarding the constitutive engagement of adhesion receptors in circulating CLL cells, and the role in this context of plasma/plasma components. To address this issue, the proteomic profiles of circulating leukemic cells from 80 CLL cases expressing or not CD49d were explored using a reverse phase protein microarray (RPMA) approach quantitatively analysing 77 proteins (61 phosphoproteins) known to be involved in translational control, cell growth, apoptosis, B cell receptor and cytoskeletal signaling. Comparison of the signaling activation portrait between 40 CD49d pos and 40 CD49d neg CLL cases highlighted the over-expression in CD49d pos CLL of proteins involved in the regulation of integrin-mediated cytoskeletal dynamics, such as phospho-p21-activated kinase (PAK1 Ser199-204/PAK2 Ser192-197; p=0.0005), phospho-LIM kinase (LIMK1 Thr508/LIMK2 Thr505; p=0.00001) and the adaptor protein CrkII Tyr221 (p=0.039). Moreover, CD49d pos CLL cells were characterized by a high correlation between proteins involved in integrin-mediated signal trasduction, including the focal-adhesion kinase (FAK Tyr576-Ser577), the tyrosine kinase Src Tyr527 and the adaptor protein CrkL Tyr207. Since PAK and LIMK represent key players in the modulation of the structure and the activity of actin cytoskeleton, we focused on these proteins for validation experiments. The constitutive pPAK and pLIMK overexpression in the CD49d pos CLL group was confirmed by western blot analysis comparing purified CLL cells from 3 CD49d pos versus 3 CD49d neg cases (mean fold increase >100 for both proteins). The results obtained suggest that integrin signalling is constitutively active in CD49d-expressing circulating CLL cells, pointing to a constitutive receptor engagement by CD49d ligands allegedly present in plasma. To test whether plasma constituents could modulate integrin-signaling proteins, CLL cells from 5 cases, expressing or not CD49d, were challenged with autologous plasma (1:3 dilution) for 30 seconds, 1 and 3 minutes. The presence of plasma induced a strong increase of PAK and LIMK phosphorylation intensities in CD49d pos CLL cells, starting at 30 seconds upon stimulation (mean fold increase >10 as compared to control for both proteins), and increasing after 1 and 3 minutes (mean fold increase >40 and >50 for both the proteins, respectively). Conversely, plasma stimulation did not induce pPAK and pLIMK expression modulation in CD49d neg CLL cells. Of note, pretreatment of CD49d-expressing CLL cells with the anti-CD49d HP1/2 blocking antibody, resulted in lower up-regulation of pPAK and pLIMK with an overall 60% inhibition for both the proteins (p=0.01), confirming the involvement of CD49d triggering in the observed activated signaling. Given the above results, we investigated plasma from 24 CLL patients, expressing CD49d at different levels, for the presence of the CD49d ligand fibronectin using an ELISA detection kit. All samples tested showed more than 1 mg/ml fibronectin concentration, without differences between CD49d-expressing and CD49d neg CLL cases. Altogether these results sustain the hypothesis of an active role of plasma/plasma components in the activation of CD49d-mediated integrin pathway, thus favoring the delivery of chemoresistance/pro-survival signals even in the context of circulating CLL cells. Our results may be of interest in the perspective of novel therapies (e.g. Bruton tyrosine kinase inhibitors) known to provoke a massive egress of neoplastic cells from tissue sites into the blood stream. Disclosures: No relevant conflicts of interest to declare.