In chronic lymphocytic leukemia (CLL) the distribution and prognostic impact of genetic and molec... more In chronic lymphocytic leukemia (CLL) the distribution and prognostic impact of genetic and molecular markers has been assessed on retrospective series of patients in different phases of the disease. Our aim was to assess the distribution and clinical significance of a comprehensive panel of clinical and biologic parameters prospectively evaluated at presentation in all young patients diagnosed with CLL at our Institution, taking advantage of the fact that in Italy individuals with a lymphocytosis are referred to the hematologist for the diagnostic work-up. From November 2002 to June 2008, 105 young CLL patients (<60 years-old) were diagnosed with CLL and included in this study. There were 56 males and 49 females, with a median age of 52 years-old. 81% were in Binet stage A, 19% in stages B/C. Rai stage 0 was recorded in 63% of patients, I/II in 31%, III/IV in 6%. The median white blood cell (WBC) count was 18.8 × 109/L (range 5.8–236.6). Prognosis was evaluated as the time from diagnosis to first treatment (TFI, treatment-free interval), since only 3 patients died after a median follow-up of 32.4 months (range 1 to 88). The median TFI was 43.9 months. Clinical features included: gender, WBC count, Binet and Rai stage. Serological and biologic parameters included: beta2-microglobulin, LDH, IgG immunoglobulin levels, lymphocyte morphology, T-cell subsets, IgVH mutational status, CD38 and ZAP-70 expression, cytogenetic abnormalities evaluated by FISH, p53 protein expression and p53 gene sequencing (exon 5 to 8). The distribution of the prognostic markers is summarized in the table. Raised beta2-microglobulin and LDH were present only in 5% and 15% of cases, respectively. The CD4/CD8 ratio was normal in almost all cases. The proportion of unmutated IgVH, CD38 and ZAP-70 positive cases was about one third of the cohort of CLL patients at diagnosis. The incidence of del(17p) (cut off >20% cells) and del(11q) (cut off >10% cells) was 2% and 7%, respectively. Patients with del(17p) or del(11q) exclusively showed unmutated IgVH and ZAP-70+, and were mostly CD38+. p53 mutations were present in 4 cases, 3 with unmutated IgVH and del(17p) and 1 with mutated IgVH and no del(17p). In univariate analysis, the following variables resulted associated to a short TFI: advanced stage Binet B/C and Rai I/IV (<0.0001), WBC count (<0.0001), proportion of CD3+ cells <16% (0.0002), raised beta2-microglobulin (<0.0001) and LDH (<0.0001), unmutated IgVH (<0.0001), CD38+ (<0.0001), ZAP-70+ (<0.0018), adverse cytogenetic abnormalities (del(17p), del(11q), +12) (<0.0001). Atypical CLL morphology showed a trend for significance (0.06). Multivariate analysis on TFI - including WBC count (as continuous variable), CD3 %, LDH, IgVH mutation status, ZAP-70 and CD38 expression and corrected with interaction between WBC and IgVH status - was focused on the 84 patients with Binet stage A. High WBC count, raised LDH, unmutated IgVH resulted as unfavorable prognostic factors, whilst the proportion of CD3+ cells was associated with a better outcome. Neither ZAP-70 or CD38 showed an independent prognostic value. In CLL cases with discordant expression of ZAP-70 and IgVH mutation status (25% of cases), the latter appeared to be more relevant than ZAP-70 in determining the TFI. In conclusion, unmutated IgVH, raised LDH, WBC count and a low proportion of CD3+ cells at diagnosis are significant predictors of TFI in early stage CLL. This group represents about one third of young patients at diagnosis. Adverse FISH abnormalities are present only in a small subgroup of cases in the early phases of the disease. Young CLL patients at diagnosis N° of cases % Raised beta2-microglobulin (>3400 ng/l) 5/102 5% Raised LDH 16/105 15% Hypo IgG 21/98 21% Atypical morphology 27/104 26% CD3+ cells (<16%) 60/105 57% CD4/CD8 (<1) 3/101 3% IgVH mutated (≥98%) 67/103 65% IgVH unmutated (<98%) 36/103 35% CD38 ≥7% 25/104 24% ZAP-70 ≥10% 39/100 39% Del(17p) >20% 2/104 2% Del(11q) >10% 7/104 7% +12 >5% 7/104 7% Del(13q) >5% isolated 59/104 57% Normal (none of the above) 29/104 28% p53 protein expression 3/100 3% p53 gene mutation 4/105 4%
The IL7 receptor a chain, encoded by the IL7R gene, heterodimerizes with the IL-2Rc (common gamma... more The IL7 receptor a chain, encoded by the IL7R gene, heterodimerizes with the IL-2Rc (common gamma) chain to form the IL7 receptor, or with the cytokine receptor-like factor 2 (CRLF2) for the thymic stromal lymphopoietin (TSLP) receptor, in both B and T cells [1,2]. Signals from the IL7 and TSLP receptors activate the JAK/STAT and PI3K/Akt/mTOR pathways, and are essential for the normal development and maintenance of the immune system [3]. The IL7/IL7R axis is also crucial for leukemogenesis: in fact, IL7 transgenic mice develop lymphomas [4] and AKR/J mice – which develop spontaneous thymic lymphomas – have high IL7R levels [5]. While activating mutations in IL7R has been well described in Band T-cell acute lymphoblastic leukemia (ALL) [6], IL7R overexpression has been poorly investigated. Recently, Ge et al. [7] evaluated IL7R expression levels in a small cohort of adult Band T-ALL samples by real-time quantitative reverse-transcription polymerase-chain-reaction (Q-RT-PCR) and reported that in B-ALL, the IL7R expression levels were significantly higher than in normal bone marrow controls and that patients with IL7R overexpression displayed low SH2B3 expression levels. Conversely, in T-ALL an association between IL7R and SH2B3 expression was not found. Clinically, B-ALL patients with a high expression of IL7R and low expression of SH2B3 showed high-risk features and a lower event-free survival (EFS) compared to the cases with low IL7R and high SH2B3 expression levels. These observations prompted us to further investigate the prognostic impact of IL7R expression in a large series of adult ALL and to explore the efficacy of specific inhibitors targeting the IL7R signaling pathway. IL7R (NM_002185.3) expression levels were assessed using previously generated gene expression profile (GEP) data (HGU133 Plus 2.0, Affymetrix, Santa Clara, CA) [8] in a cohort of 265 adult acute leukemias sampled at diagnosis and enrolled in different GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) protocols. This cohort included 173 B-ALL, 66 T-ALL and, for comparison purposes, 26 acute myeloid leukemias (AML). We documented that IL7R expression levels were highly variable across the ALL samples. In B-ALL, the median expression level – computed as normalized Affymetrix units – was 308.8 (range 23.4-5557) and in T-ALL the median expression level was 680.5 (range 58.9–5874); AML samples displayed the lowest expression values (mean 158.20; range 38.76–427.8). Within the B-ALL molecular subtypes, IL7R expression values were higher in the cases negative for the recurrent fusion genes (i.e. BCR/ABL1, ETV6/RUNX1, TCF3/PBX1, KMT2A(MLL)/rearrangements, defined as BNEG throughout the text) (n1⁄4 77, median 449.8; range 43.8–3000) and in TCF3/PBX1þ cases (n1⁄4 7, median 670.5; range 448.1–1851) than in BCR/ABL1þ (n1⁄4 72, median 195.4; range 23.4–5557) and KTM2A/AFF1þ cases (n1⁄4 17, median 283.3; range 55.1–1591); a statistically significant difference in IL7R expression values was observed between B-NEG and BCR/ABL1þ cases (p1⁄4 .035). To validate the microarray results, gene expression levels of IL7R were quantified by Q-RT-PCR, as detailed in the Supplemental Material. We found a high correlation (Pearson coefficient: 0.89) between the Q-RT-PCR and microarray values, thus confirming the GEP results.
Background: Great interest has been raised recently by the design of new adoptive immunotherapeut... more Background: Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application. Materials and methods: Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing. Results: NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing. Discussion: These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.
blood cell count at diagnosis and immunoglobulin variable region gene mutations are independent p... more blood cell count at diagnosis and immunoglobulin variable region gene mutations are independent predictors of treatment-free survival in young patients with stage A chronic lymphocytic leukemia.
In chronic lymphocytic leukemia (CLL), spontaneous regressions are an exceptional phenomenon, who... more In chronic lymphocytic leukemia (CLL), spontaneous regressions are an exceptional phenomenon, whose biologic features are unknown. We describe 9 CLL patients who underwent a spontaneous clinical regression over an 11-year followup, despite a residual neoplastic clone detected by flow cytometry. CD38 and ZAP-70 were negative in all cases. Immunoglobulin heavy chain variable region (IgVH) genes, mutated in all 7 evaluable patients, were restricted to the VH3 family in 6, with the usage of V H 3-30 gene in 2. The light chain variable region genes were mutated in 6 of 8 cases, with the use of V 4-1 gene in 3. Microarray analysis of CLL cells showed a distinctive genomic profile with an overrepresentation of BCRrelated and ribosomal genes, regulators of signal transduction and transcription. The number of activated T lymphocytes expressing IFN-␥, TNF-␣, and IL-4 was similar between CLL in spontaneous regression and healthy persons. In conclusion , spontaneous clinical regressions can occur in CLL despite the persistence of the neoplastic clone, and the biologic features include negative CD38 and ZAP-70, mutated V H 3-30 and V 4-1 genes. The peculiar gene profile suggests that BCR signaling may play an important role in this scenario as the most significant feature of the leukemic clone in regression. (Blood.
The management of acute lymphoblastic leukemia (ALL) patients has witnessed profound changes in r... more The management of acute lymphoblastic leukemia (ALL) patients has witnessed profound changes in recent years. Nonetheless, most patients tend to relapse, underlining the need for new therapeutic approaches. The anti-leukemic potential of natural killer (NK) cells has over the years raised considerable interest. In this study, we developed an efficient method for the expansion and activation of NK cells isolated from healthy donors and ALL patients for clinical use. NK cell products were derived from peripheral blood mononuclear cells of 35 healthy donors and 4 B-lineage ALL by immunomagnetic CD3 T cell depletion followed by CD56 cell enrichment. Isolated NK cells were expanded and stimulated in serum-free medium supplemented with irradiated autologous feeder cells and autologous plasma in the presence of clinical grade interleukin (IL)-2 and IL-15 for 14 days. Healthy donor NK cells expanded on average 34.9 ± 10.4 fold and were represented, after expansion, by a highly pure population of CD3(-)CD56(+) cells showing a significant upregulation of natural cytotoxicity receptors, activating receptors and maturation markers. These expanded effectors showed cytolytic activity against K562 cells and, most importantly, against primary adult B-lineage ALL blasts. NK cells could be efficiently isolated and expanded-on average 39.5 ± 20.3 fold-also from primary B-lineage ALL samples of patients in complete remission. The expanded NK cells from these patients showed a significantly increased expression of the NKG2D- and DNAM1-activating receptors and were cytotoxic against K562 cells. These data provide the basis for developing new immunotherapeutic strategies for the management of ALL patients.
MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that modulate the expression of gene... more MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that modulate the expression of genes at the posttranscriptional level. These small molecules have been shown to be involved in cancer, apoptosis, and cell metabolism. In the present study we provide an informative profile of the expression of miRNAs in primary chronic lymphocytic leukemia (CLL) cells using 2 independent and quantitative methods: miRNA cloning and quantitative real-time-polymerase chain reaction (qRT-PCR) of mature miRNAs. Both approaches show that miR-21 and miR-155 are dramatically overexpressed in patients with CLL, although the corresponding genomic loci are not amplified. miR-150 and miR-92 are also significantly deregulated in patients with CLL. In addition, we detected a marked miR-15a and miR-16 decrease in about 11% of cases. Finally, we identified a set of miRNAs whose expression correlates with biologic parameters of prognostic relevance, particularly with the mutational status of the IgV H genes. In summary, the results of this study offer for the first time a comprehensive and quantitative profile of miRNA expression in CLL and their healthy counterpart, suggesting that miRNAs could play a primary role in the disease itself.
Imatinib mesylate (Imatinib) is a potent inhibitor of defined tyrosine kinases and is effectively... more Imatinib mesylate (Imatinib) is a potent inhibitor of defined tyrosine kinases and is effectively used for the treatment of malignancies characterized by the constitutive activation of these tyrosine kinases, such as Philadelphia chromosome-positive (Ph ?) leukemias and gastrointestinal stromal tumors. Suppressive as well as stimulating effects of this drug on T lymphocytes or dendritic cells (DC), which play a major role in immune tumor surveillance, have been reported. For this reason, we questioned whether Imatinib could also affect the phenotypic and functional properties of these subpopulations in Ph ? acute lymphoblastic leukemia (ALL) patients on prolonged Imatinib maintenance treatment. Circulating T lymphocytes and NK cells from Imatinib-treated Ph ? ALL patients showed a subset distribution comparable to that of healthy donors. In addition, T-cell immunomodulant cytokine production (IFN-c, TNF-a) and proliferative responses were not impaired. A normal monocyte-derived DC differentiation and apoptotic body loading capacity was also observed in the majority of Imatinibtreated patients. In contrast, an impairment in the DC intracellular production of IL-12 was recorded, although this was not observed when normal DC were exposed in vitro to Imatinib. Finally, in vivo Imatinib treatment did not affect the T-lymphocyte proliferation and IFN-c production induced by leukemic apoptotic body-loaded DC, underling the potential capability of these cells to generate a specific immune response against tumoral antigens. Taken together, these findings provide evidence that immunotherapeutic approaches aimed at controlling residual disease in Ph ? ALL patients in hematologic remission are not jeopardized by the long-term administration of Imatinib.
TP53-disrupted chronic lymphocytic leukaemia (CLL) patients show a suboptimal long-term response ... more TP53-disrupted chronic lymphocytic leukaemia (CLL) patients show a suboptimal long-term response to ibrutinib. We hereby report that ibrutinib-induced in vitro apoptosis and proliferation inhibition were significantly lower in TP53-mutated (TP53-M) CLL cells compared to TP53 wild-type cells. Contrariwise, venetoclax effectively killed TP53-M cells. Gene expression profile analysis of TP53-M cells revealed a downmodulation of B-cell receptor (BCR)-related genes and an upmodulation of genes with anti-apoptotic/pro-survival activity, suggesting that the survival and proliferation of TP53-M cells are less dependent on the BCR pathway. These observations further support the use of drug combinations for the optimal management of TP53-M CLL patients.
Background: Chronic lymphocytic leukemia (CLL) has a heterogeneous clinical course. Beside patien... more Background: Chronic lymphocytic leukemia (CLL) has a heterogeneous clinical course. Beside patients requiring immediate treatment, others show an initial indolent phase followed by progression and others do not progress for decades. The latter two subgroups usually display mutated IGHV genes and a favorable FISH profile. Patients and methods: Patients with absence of disease progression for over 10 years (10-34) from diagnosis were defined as ultra-stable CLL (US-CLL). Forty US-CLL underwent extensive characterization including whole exome sequencing (WES), ultradeep sequencing and copy number aberration (CNA) analysis to define their unexplored genetic landscape. Microarray analysis, comparing US-CLL with non-US-CLL with similar immunogenetic features (mutated IGHV/favorable FISH), was also carried out to recognize US-CLL at diagnosis. Results: WES was carried out in 20 US-CLL and 84 non-silent somatic mutations in 78 genes were found. When re-tested in a validation cohort of 20 further US-CLL, no recurrent lesion was identified. No clonal mutations of NOTCH1, BIRC3, SF3B1 and TP53 were found, including ATM and other potential progression driving mutations. CNA analysis identified 31 lesions, none with known poor prognostic impact. No novel recurrent lesion was identified: most cases showed no lesions (38%) or an isolated del(13q) (31%). The expression of 6 genes, selected from a gene expression profile analysis by microarray and quantified by droplet digital PCR on a cohort of 79 CLL (58 US-CLL and 21 non-US-CLL), allowed to build a decision-tree capable of recognizing at diagnosis US-CLL patients. Conclusions: The genetic landscape of US-CLL is characterized by the absence of known unfavorable driver mutations/ CNA and of novel recurrent genetic lesions. Among CLL patients with favorable immunogenetics, a decision-tree based on the expression of 6 genes may identify at diagnosis patients who are likely to maintain an indolent disease for decades.
the pathways of NK cell/ALL blast interaction still need to be better clarified. In this study, w... more the pathways of NK cell/ALL blast interaction still need to be better clarified. In this study, we addressed the pathways of NK cell/ALL blast recognition and compared the expression of activating ligands among pediatric and adult ALL patients. Molecularly-defined subgroups of ALL patients were considered in the analysis, and functional tests were performed to confirm the results of the phenotypic findings. Methods Patients A total of 103 newly diagnosed cases of ALL, including 46 adults (median age 34 years; range 18-74 years) and 57 children (median age 4 years; range 0.1-17 years) were investigated between December 2011 and February 2013. Based on the immunophenotypic profile, the case series was subdivided as follows: 90 B-lineage ALL (B-ALL) (39 adult and 51 pediatric cases) and 13 T-lineage ALL (T-ALL) (7 adult and 6 pediatric cases). Within the adult BALL cohort, the following fusion transcripts were identified: BCR-ABL in 15 cases, MLL-AF4 in 7, E2A-PBX1 in 2, while the remaining 15 cases were negative for the most common molecular lesions found in ALL. In pediatric BALL , the BCR-ABL transcript was found in 6 patients, MLL-AF4 in 3, MLL-ENL in 1, TEL-AML1 in 10; 31 patients did not show any of the molecular aberrations investigated. Informed consent for biological studies was obtained from patients or their legal guardians in accordance with the Declaration of Helsinki. The study was approved by the local ethics committee.
Chronic lymphocytic leukemia (CLL) presents a highly variable clinical course, with some patients... more Chronic lymphocytic leukemia (CLL) presents a highly variable clinical course, with some patients surviving for many years without requiring treatment, while others have rapidly progressing disease, require aggressive treatment and show a short life expectancy. Several biological features of the leukemic cells - the mutational status of the immunoglobulin (Ig) heavy (H) chain variable (V) genes, cytogenetic alterations, ZAP-70 and CD38 antigen expression, p53 dysfunctions – bear prognostic value and allow to stratify patients into risk categories. The deletion of the chromosome region 11q22–q23 that occurs in 10–20% of cases represents the second most common genetic abnormality in CLL and defines a subgroup of patients characterized by advanced progressive disease and poor survival. The ATM (ataxia telangiectasia-mutated) gene is located in this region and several studies suggest that the 11q deletion results in ATM gene inactivation. The ATM gene consists of 66 exons, 62 (5–66) of which are coding exons. The ATM protein is a nuclear serine/threonine kinase of 350 kDa induced by chromosomal double-strand breaks that arise endogenously or after exposure to DNA-damaging agents, including ionizing radiations and drugs. It protects the integrity of the genome in several ways: it regulates the arrest of the cell cycle at the G1/S and G2/M phases to prevent the processing of damaged DNA; it activates DNA repair pathways and induces apoptosis if the DNA damage cannot be repaired. In the present study we examined the status of the ATM gene by DHPLC (Denaturing High Performance Liquid Cromatography) in a series of untreated CLL patients studied at the Division of Hematology, “Sapienza” University of Rome. In addition, a multiplex gene dosage analysis of the ATM gene was performed by MLPA (Multiplex Ligation Probe Amplification) in all patients. The results obtained have been correlated with the molecular status of the IgVH genes, the presence of cytogenetic abnormalities, ZAP-70 and CD38 expression, p53 mutations, and with the gene expression profile. Mutational screening of the ATM gene identified mutations in 8 of the 57 CLL patients studied (14%): 6 missense, 8095C>T (Pro2699Ser), 8071C>T (Arg2691Cys), 2476A>C (Ile826Leu) and, in 3 patients, 1435G>T (Asp479Tyr); 1 frameshift: 2502insA; 1 splicing mutation: IVS29+5G>A. In addition, 14 different variants and polymorphisms of unknown functional significance were found. The 8 ATM mutated cases showed the following features: 6 were IgVH unmutated, 4 were ZAP70+, 5 were CD38+, 4 harbored a 13q14 deletion, only 1 patient showed a 17p13 deletion and 2 a 14q32 deletion; furthermore, 2/8 patients showed a p53 gene mutation; none showed the 11q deletion. MLPA analysis identified 6 patients with 11q deletion and all of them showed at least 20% of cells positive for the 11q23 deletion evaluated by FISH; only 1 case showed a 17p13 deletion. In these cases, the IgVH gene was always unmutated and ZAP70 was positive; 4 cases were also CD38+. ATM gene mutated and deleted CLL cases showed a peculiar gene profile characterized by the up-regulation and down-regulation of genes involved in apoptosis and DNA repair function. On the contrary, no gene profile difference was found in patients carrying ATM gene polymorphisms. Patients with ATM gene mutated and deleted showed a short time to treatment, 23 and 19 months respectively. The results of this study indicate that ATM gene alterations - mutations or deletions - account for 24% of untreated CLL patients, are associated with a defined gene profile and are characterized by a poor prognosis. It is important to underline that these patients have been evaluated before any treatment and that ATM mutations have been found in the absence of 11q23 deletions. ATM alterations appear to represent the most frequent genetic abnormality with unfavorable prognostic implications in CLL and should be investigated prior to initiating therapy in order to plan an appropriate treatment algorithm.
CD4+CD25+ T-regulatory (Treg) cells have been shown to regulate self and allograft tolerance and ... more CD4+CD25+ T-regulatory (Treg) cells have been shown to regulate self and allograft tolerance and to suppress allogeneic immune response. Studies in mouse models of bone marrow (BM) transplantation have shown that the infusion of culture-expanded Tregs can be effective in preventing and suppressing graft-versus-host disease (GVHD), while still allowing a graft-versus-leukemia effect (GVL). Cord blood (CB) represents the most recent source of hematopietic stem cell transplantation (HSCT) used in the unrelated transplant setting. Comparative studies have shown that unrelated CB transplant is an acceptable alternative to unrelated BM transplantation, characterized by a lower risk of transplant-related mortality due to GVHD. CB has been reported to contain Tregs, but minimal data are available on these cells. Aim of this study was to compare the suppressive functions of Tregs obtained from CB units with those obtained from the peripheral blood (PB) of patients who have undergone an allogeneic BM transplantation and of normal donors. Treg cells were purified from mononuclear cells obtained from CB units or PB using the CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and expanded for 6 days in 96-well U-Bottom plates coated with anti-CD3 and anti-CD28 MoAbs plus IL-2. To assess the suppressive functions of Treg cells, expanded CD4+CD25+ Tregs from CB units or PB were seeded with naïve autologous effector CD4+CD25- T cells stimulated with allogeneic dendritic cells (DC) pulsed with apoptotic leukemic blasts, then incubated with [3H]-thymidine and counted in a beta-counter. Suppressor activity was measured as [3H]-thymidine incorporation in the presence or absence of Tregs. The immunophenotypic analysis of Tregs from CB units - in terms of expression of surface CD4, CD25, CD62L, cytoplasmic CTLA-4 and Foxp3 - before and after expansion, did not show any specific peculiarity. CB units (n = 6) contained a proportion of Tregs comparable to those contained in the PB of allotransplanted patients (n = 8) or in the PB of normal donors (n = 8) (mean % ± SD, CB 0.28% ± 0.41; PB patients 0.4% ± 0.4; PB donors CB 0.45% ± 0.29). On the contrary, Tregs from CB units and from the PB of allotransplanted patients showed a higher expansion capacity when compared to Tregs from the PB of normal donors (mean fold increase ± SD, CB units 11.5 ± 9.0; PB patients 9.6 ± 5.0; PB donors CB 5.2 ± 2.7). Preliminary data also show that Tregs expanded from CB units (n = 2) exert a higher suppressive function on the proliferative reaction of T cells stimulated by allogeneic DC if compared to Tregs expanded from the PB of allotransplanted patients (n = 2) (mean fold reduction ± SD, CB 5.25 ± 3.7; PB patients 3.8 ± 3.3). These results demonstrate that Treg cells contained in CB units present an expansion capacity superior to that of Tregs from PB of normal donors; moreover, these expanded cells seem to exert a potent suppressive function of the proliferative effect in mixed lymphocyte reaction culture assays. These data offer further insights in the understanding of the biology of CB transplantation and may indicate a possible clinical use of expanded Tregs for the prevention and management of GVHD in the setting of CB transplantation.
The capacity to generate effective dendritic cells (DC) from adult acute lymphoblastic leukemia (... more The capacity to generate effective dendritic cells (DC) from adult acute lymphoblastic leukemia (ALL) patients in complete remission (CR) and off-therapy was investigated. Monocyte-derived DC cultured in the presence of granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4 and tumor necrosis factor (TNF)-alpha expressed maturation markers, produced IL-12 and loaded apoptotic bodies to a similar extent to normal DC. Patients' circulating T and NK lymphocytes were normally represented and, after stimulation, were capable of producing TNF-alpha and interferon-gamma to a similar extent to control lymphocytes. DC loaded with leukemia-derived apoptotic bodies increased their ability to stimulate both allogeneic and autologous lymphocytes, and to generate specific anti-leukemic CD3 + cells. These findings offer a rationale for the design of DC-based vaccine programs for adult ALL patients in CR with the aim of controlling/eradicating the disease.
The immunologic reconstitution is ultimately responsible of the clinical outcome of patients who ... more The immunologic reconstitution is ultimately responsible of the clinical outcome of patients who have undergone an allogeneic stem cell transplantation (SCT). The occurrence of graft-versus-host disease (GVHD), which represents the major cause of morbidity and mortality after the transplant correlates with the concentration in the peripheral blood (PB) of regulatory T cells (Tregs). In this study we aim at demonstrating that not only the concentration but also the functional capacities and the degree of activity of Tregs act as an important regulator of alloreactivity and may help to predict the risk of acute and chronic GVHD in the post-transplant period. Sixteen patients who underwent an allogeneic SCT were evaluated at 1 year from transplant. Tregs were expanded from the PB of these patients and from 8 normal donors; their expansion capacity, phenotype, suppressor activity and IL-10 production were measured. Tregs expanded from patients without GVHD exerted a higher suppressive function on the proliferative reaction of T cells and showed a higher IL-10 production capacity compared to patients with acute or chronic GVHD. These results document that the functional activity and the suppressor capacity of Tregs after an allogeneic SCT may protect from GVHD, and support the design of clinical protocols based on the infusion of expanded and activated Tregs.
Background: CLL is characterized by an heterogeneous clinical course, with some patients living m... more Background: CLL is characterized by an heterogeneous clinical course, with some patients living many years without therapy and other patients presenting an aggressive disease. Different biologic properties of the leukemic cells have important prognostic implications. In particular, the IgVH gene mutational status, chromosomal abnormalities, expression of CD38 and ZAP-70 have been correlated with the clinical course of the disease. There is also growing evidence that angiogenesis may be linked to the pathogenesis and outcome of CLL. In particular, vascular endothelial growth factor A (VEGF-A) has been detected in the serum and bone marrow biopsy cells analyzed by immunostaining: both events have been correlated with a poor prognostic likelihood. Aims: The goal of this study was to obtain a more complete characterization of the angiogenic potential of CLL cells, to correlate the angiogenic capacity with other prognostic parameters and to investigate the possibility of overcoming this property by specific VEGF-signaling inhibitors. Methods: Eighteen patients with a diagnosis of CLL, based on morphologic and immunophenotypic criteria, have been evaluated for the expression of CD38 and ZAP-70, and for the IgVH gene mutational status. An Angiokit test (TCS Cell Works) was utilized to evaluate the capacity of purified CD19+ CLL cells to stimulate the growth and tubule formation of human endothelial cells and the relative roles of an anti-VEGF specific antibody and of PTK787/ZK 222584 (PTK/ZK, Schering, Novartis), a novel, oral angiogenesis and lymphangiogenesis inhibitor that blocks tyrosine kinase signaling from all known VEGFRs. The quantification of endothelial tubule formation was carried out using a specific software. After stimulation with a biotinylated goat anti-human IgM F(ab)2, leukemia cells were evaluated for gene expression profiling using the Affymetrix HGU133 Plus 2.0 gene chip. Results: In 9 patients, the leukemic cells were CD38+, ZAP-70+ and showed an unmutated IgVH status; in the remaining 9, the leukemic cells were CD38−, ZAP-70− and with a mutated IgVH status. In CLL patients with poor prognostic features - CD38+, ZAP-70+ and unmutated IgVH - purified leukemic cells showed a significantly greater capacity to stimulate the growth and tubule formation (3407 ± 968 tubules) of human endothelial cells compared to leukemic cells obtained from CLL patients with good prognostic factors, i.e. CD38−, ZAP-70− and mutated IgVH, (1550 ± 624 tubules). This property was amplified following sIgM cross-linking, that resulted in the upregulation of angiogenetic genes, including: EGR-1, SDF2L1, TNFAIP3, PTGER4, FIBP, NR4A3. A reduction of VEGF-A-induced tubule number was observed when leukemic cells were cultured with the anti-VEGF antibody (43–82% of inhibition) and, to a further extent, with the PTK/ZK inhibitor (90–96% of inhibition) compared to purified CLL cells alone. Conclusions: In CLL patients with poor prognostic factors, leukemic cells display an angiogenic capability significantly higher than that of cells from patients with favorable prognostic factors. The current findings provide the rationale for investigating the potential clinical role of anti-angiogenic agents, such as PTK/ZK, in the management of CLL patients.
In chronic lymphocytic leukemia (CLL) the distribution and prognostic impact of genetic and molec... more In chronic lymphocytic leukemia (CLL) the distribution and prognostic impact of genetic and molecular markers has been assessed on retrospective series of patients in different phases of the disease. Our aim was to assess the distribution and clinical significance of a comprehensive panel of clinical and biologic parameters prospectively evaluated at presentation in all young patients diagnosed with CLL at our Institution, taking advantage of the fact that in Italy individuals with a lymphocytosis are referred to the hematologist for the diagnostic work-up. From November 2002 to June 2008, 105 young CLL patients (<60 years-old) were diagnosed with CLL and included in this study. There were 56 males and 49 females, with a median age of 52 years-old. 81% were in Binet stage A, 19% in stages B/C. Rai stage 0 was recorded in 63% of patients, I/II in 31%, III/IV in 6%. The median white blood cell (WBC) count was 18.8 × 109/L (range 5.8–236.6). Prognosis was evaluated as the time from diagnosis to first treatment (TFI, treatment-free interval), since only 3 patients died after a median follow-up of 32.4 months (range 1 to 88). The median TFI was 43.9 months. Clinical features included: gender, WBC count, Binet and Rai stage. Serological and biologic parameters included: beta2-microglobulin, LDH, IgG immunoglobulin levels, lymphocyte morphology, T-cell subsets, IgVH mutational status, CD38 and ZAP-70 expression, cytogenetic abnormalities evaluated by FISH, p53 protein expression and p53 gene sequencing (exon 5 to 8). The distribution of the prognostic markers is summarized in the table. Raised beta2-microglobulin and LDH were present only in 5% and 15% of cases, respectively. The CD4/CD8 ratio was normal in almost all cases. The proportion of unmutated IgVH, CD38 and ZAP-70 positive cases was about one third of the cohort of CLL patients at diagnosis. The incidence of del(17p) (cut off >20% cells) and del(11q) (cut off >10% cells) was 2% and 7%, respectively. Patients with del(17p) or del(11q) exclusively showed unmutated IgVH and ZAP-70+, and were mostly CD38+. p53 mutations were present in 4 cases, 3 with unmutated IgVH and del(17p) and 1 with mutated IgVH and no del(17p). In univariate analysis, the following variables resulted associated to a short TFI: advanced stage Binet B/C and Rai I/IV (<0.0001), WBC count (<0.0001), proportion of CD3+ cells <16% (0.0002), raised beta2-microglobulin (<0.0001) and LDH (<0.0001), unmutated IgVH (<0.0001), CD38+ (<0.0001), ZAP-70+ (<0.0018), adverse cytogenetic abnormalities (del(17p), del(11q), +12) (<0.0001). Atypical CLL morphology showed a trend for significance (0.06). Multivariate analysis on TFI - including WBC count (as continuous variable), CD3 %, LDH, IgVH mutation status, ZAP-70 and CD38 expression and corrected with interaction between WBC and IgVH status - was focused on the 84 patients with Binet stage A. High WBC count, raised LDH, unmutated IgVH resulted as unfavorable prognostic factors, whilst the proportion of CD3+ cells was associated with a better outcome. Neither ZAP-70 or CD38 showed an independent prognostic value. In CLL cases with discordant expression of ZAP-70 and IgVH mutation status (25% of cases), the latter appeared to be more relevant than ZAP-70 in determining the TFI. In conclusion, unmutated IgVH, raised LDH, WBC count and a low proportion of CD3+ cells at diagnosis are significant predictors of TFI in early stage CLL. This group represents about one third of young patients at diagnosis. Adverse FISH abnormalities are present only in a small subgroup of cases in the early phases of the disease. Young CLL patients at diagnosis N° of cases % Raised beta2-microglobulin (>3400 ng/l) 5/102 5% Raised LDH 16/105 15% Hypo IgG 21/98 21% Atypical morphology 27/104 26% CD3+ cells (<16%) 60/105 57% CD4/CD8 (<1) 3/101 3% IgVH mutated (≥98%) 67/103 65% IgVH unmutated (<98%) 36/103 35% CD38 ≥7% 25/104 24% ZAP-70 ≥10% 39/100 39% Del(17p) >20% 2/104 2% Del(11q) >10% 7/104 7% +12 >5% 7/104 7% Del(13q) >5% isolated 59/104 57% Normal (none of the above) 29/104 28% p53 protein expression 3/100 3% p53 gene mutation 4/105 4%
The IL7 receptor a chain, encoded by the IL7R gene, heterodimerizes with the IL-2Rc (common gamma... more The IL7 receptor a chain, encoded by the IL7R gene, heterodimerizes with the IL-2Rc (common gamma) chain to form the IL7 receptor, or with the cytokine receptor-like factor 2 (CRLF2) for the thymic stromal lymphopoietin (TSLP) receptor, in both B and T cells [1,2]. Signals from the IL7 and TSLP receptors activate the JAK/STAT and PI3K/Akt/mTOR pathways, and are essential for the normal development and maintenance of the immune system [3]. The IL7/IL7R axis is also crucial for leukemogenesis: in fact, IL7 transgenic mice develop lymphomas [4] and AKR/J mice – which develop spontaneous thymic lymphomas – have high IL7R levels [5]. While activating mutations in IL7R has been well described in Band T-cell acute lymphoblastic leukemia (ALL) [6], IL7R overexpression has been poorly investigated. Recently, Ge et al. [7] evaluated IL7R expression levels in a small cohort of adult Band T-ALL samples by real-time quantitative reverse-transcription polymerase-chain-reaction (Q-RT-PCR) and reported that in B-ALL, the IL7R expression levels were significantly higher than in normal bone marrow controls and that patients with IL7R overexpression displayed low SH2B3 expression levels. Conversely, in T-ALL an association between IL7R and SH2B3 expression was not found. Clinically, B-ALL patients with a high expression of IL7R and low expression of SH2B3 showed high-risk features and a lower event-free survival (EFS) compared to the cases with low IL7R and high SH2B3 expression levels. These observations prompted us to further investigate the prognostic impact of IL7R expression in a large series of adult ALL and to explore the efficacy of specific inhibitors targeting the IL7R signaling pathway. IL7R (NM_002185.3) expression levels were assessed using previously generated gene expression profile (GEP) data (HGU133 Plus 2.0, Affymetrix, Santa Clara, CA) [8] in a cohort of 265 adult acute leukemias sampled at diagnosis and enrolled in different GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) protocols. This cohort included 173 B-ALL, 66 T-ALL and, for comparison purposes, 26 acute myeloid leukemias (AML). We documented that IL7R expression levels were highly variable across the ALL samples. In B-ALL, the median expression level – computed as normalized Affymetrix units – was 308.8 (range 23.4-5557) and in T-ALL the median expression level was 680.5 (range 58.9–5874); AML samples displayed the lowest expression values (mean 158.20; range 38.76–427.8). Within the B-ALL molecular subtypes, IL7R expression values were higher in the cases negative for the recurrent fusion genes (i.e. BCR/ABL1, ETV6/RUNX1, TCF3/PBX1, KMT2A(MLL)/rearrangements, defined as BNEG throughout the text) (n1⁄4 77, median 449.8; range 43.8–3000) and in TCF3/PBX1þ cases (n1⁄4 7, median 670.5; range 448.1–1851) than in BCR/ABL1þ (n1⁄4 72, median 195.4; range 23.4–5557) and KTM2A/AFF1þ cases (n1⁄4 17, median 283.3; range 55.1–1591); a statistically significant difference in IL7R expression values was observed between B-NEG and BCR/ABL1þ cases (p1⁄4 .035). To validate the microarray results, gene expression levels of IL7R were quantified by Q-RT-PCR, as detailed in the Supplemental Material. We found a high correlation (Pearson coefficient: 0.89) between the Q-RT-PCR and microarray values, thus confirming the GEP results.
Background: Great interest has been raised recently by the design of new adoptive immunotherapeut... more Background: Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application. Materials and methods: Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing. Results: NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing. Discussion: These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.
blood cell count at diagnosis and immunoglobulin variable region gene mutations are independent p... more blood cell count at diagnosis and immunoglobulin variable region gene mutations are independent predictors of treatment-free survival in young patients with stage A chronic lymphocytic leukemia.
In chronic lymphocytic leukemia (CLL), spontaneous regressions are an exceptional phenomenon, who... more In chronic lymphocytic leukemia (CLL), spontaneous regressions are an exceptional phenomenon, whose biologic features are unknown. We describe 9 CLL patients who underwent a spontaneous clinical regression over an 11-year followup, despite a residual neoplastic clone detected by flow cytometry. CD38 and ZAP-70 were negative in all cases. Immunoglobulin heavy chain variable region (IgVH) genes, mutated in all 7 evaluable patients, were restricted to the VH3 family in 6, with the usage of V H 3-30 gene in 2. The light chain variable region genes were mutated in 6 of 8 cases, with the use of V 4-1 gene in 3. Microarray analysis of CLL cells showed a distinctive genomic profile with an overrepresentation of BCRrelated and ribosomal genes, regulators of signal transduction and transcription. The number of activated T lymphocytes expressing IFN-␥, TNF-␣, and IL-4 was similar between CLL in spontaneous regression and healthy persons. In conclusion , spontaneous clinical regressions can occur in CLL despite the persistence of the neoplastic clone, and the biologic features include negative CD38 and ZAP-70, mutated V H 3-30 and V 4-1 genes. The peculiar gene profile suggests that BCR signaling may play an important role in this scenario as the most significant feature of the leukemic clone in regression. (Blood.
The management of acute lymphoblastic leukemia (ALL) patients has witnessed profound changes in r... more The management of acute lymphoblastic leukemia (ALL) patients has witnessed profound changes in recent years. Nonetheless, most patients tend to relapse, underlining the need for new therapeutic approaches. The anti-leukemic potential of natural killer (NK) cells has over the years raised considerable interest. In this study, we developed an efficient method for the expansion and activation of NK cells isolated from healthy donors and ALL patients for clinical use. NK cell products were derived from peripheral blood mononuclear cells of 35 healthy donors and 4 B-lineage ALL by immunomagnetic CD3 T cell depletion followed by CD56 cell enrichment. Isolated NK cells were expanded and stimulated in serum-free medium supplemented with irradiated autologous feeder cells and autologous plasma in the presence of clinical grade interleukin (IL)-2 and IL-15 for 14 days. Healthy donor NK cells expanded on average 34.9 ± 10.4 fold and were represented, after expansion, by a highly pure population of CD3(-)CD56(+) cells showing a significant upregulation of natural cytotoxicity receptors, activating receptors and maturation markers. These expanded effectors showed cytolytic activity against K562 cells and, most importantly, against primary adult B-lineage ALL blasts. NK cells could be efficiently isolated and expanded-on average 39.5 ± 20.3 fold-also from primary B-lineage ALL samples of patients in complete remission. The expanded NK cells from these patients showed a significantly increased expression of the NKG2D- and DNAM1-activating receptors and were cytotoxic against K562 cells. These data provide the basis for developing new immunotherapeutic strategies for the management of ALL patients.
MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that modulate the expression of gene... more MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that modulate the expression of genes at the posttranscriptional level. These small molecules have been shown to be involved in cancer, apoptosis, and cell metabolism. In the present study we provide an informative profile of the expression of miRNAs in primary chronic lymphocytic leukemia (CLL) cells using 2 independent and quantitative methods: miRNA cloning and quantitative real-time-polymerase chain reaction (qRT-PCR) of mature miRNAs. Both approaches show that miR-21 and miR-155 are dramatically overexpressed in patients with CLL, although the corresponding genomic loci are not amplified. miR-150 and miR-92 are also significantly deregulated in patients with CLL. In addition, we detected a marked miR-15a and miR-16 decrease in about 11% of cases. Finally, we identified a set of miRNAs whose expression correlates with biologic parameters of prognostic relevance, particularly with the mutational status of the IgV H genes. In summary, the results of this study offer for the first time a comprehensive and quantitative profile of miRNA expression in CLL and their healthy counterpart, suggesting that miRNAs could play a primary role in the disease itself.
Imatinib mesylate (Imatinib) is a potent inhibitor of defined tyrosine kinases and is effectively... more Imatinib mesylate (Imatinib) is a potent inhibitor of defined tyrosine kinases and is effectively used for the treatment of malignancies characterized by the constitutive activation of these tyrosine kinases, such as Philadelphia chromosome-positive (Ph ?) leukemias and gastrointestinal stromal tumors. Suppressive as well as stimulating effects of this drug on T lymphocytes or dendritic cells (DC), which play a major role in immune tumor surveillance, have been reported. For this reason, we questioned whether Imatinib could also affect the phenotypic and functional properties of these subpopulations in Ph ? acute lymphoblastic leukemia (ALL) patients on prolonged Imatinib maintenance treatment. Circulating T lymphocytes and NK cells from Imatinib-treated Ph ? ALL patients showed a subset distribution comparable to that of healthy donors. In addition, T-cell immunomodulant cytokine production (IFN-c, TNF-a) and proliferative responses were not impaired. A normal monocyte-derived DC differentiation and apoptotic body loading capacity was also observed in the majority of Imatinibtreated patients. In contrast, an impairment in the DC intracellular production of IL-12 was recorded, although this was not observed when normal DC were exposed in vitro to Imatinib. Finally, in vivo Imatinib treatment did not affect the T-lymphocyte proliferation and IFN-c production induced by leukemic apoptotic body-loaded DC, underling the potential capability of these cells to generate a specific immune response against tumoral antigens. Taken together, these findings provide evidence that immunotherapeutic approaches aimed at controlling residual disease in Ph ? ALL patients in hematologic remission are not jeopardized by the long-term administration of Imatinib.
TP53-disrupted chronic lymphocytic leukaemia (CLL) patients show a suboptimal long-term response ... more TP53-disrupted chronic lymphocytic leukaemia (CLL) patients show a suboptimal long-term response to ibrutinib. We hereby report that ibrutinib-induced in vitro apoptosis and proliferation inhibition were significantly lower in TP53-mutated (TP53-M) CLL cells compared to TP53 wild-type cells. Contrariwise, venetoclax effectively killed TP53-M cells. Gene expression profile analysis of TP53-M cells revealed a downmodulation of B-cell receptor (BCR)-related genes and an upmodulation of genes with anti-apoptotic/pro-survival activity, suggesting that the survival and proliferation of TP53-M cells are less dependent on the BCR pathway. These observations further support the use of drug combinations for the optimal management of TP53-M CLL patients.
Background: Chronic lymphocytic leukemia (CLL) has a heterogeneous clinical course. Beside patien... more Background: Chronic lymphocytic leukemia (CLL) has a heterogeneous clinical course. Beside patients requiring immediate treatment, others show an initial indolent phase followed by progression and others do not progress for decades. The latter two subgroups usually display mutated IGHV genes and a favorable FISH profile. Patients and methods: Patients with absence of disease progression for over 10 years (10-34) from diagnosis were defined as ultra-stable CLL (US-CLL). Forty US-CLL underwent extensive characterization including whole exome sequencing (WES), ultradeep sequencing and copy number aberration (CNA) analysis to define their unexplored genetic landscape. Microarray analysis, comparing US-CLL with non-US-CLL with similar immunogenetic features (mutated IGHV/favorable FISH), was also carried out to recognize US-CLL at diagnosis. Results: WES was carried out in 20 US-CLL and 84 non-silent somatic mutations in 78 genes were found. When re-tested in a validation cohort of 20 further US-CLL, no recurrent lesion was identified. No clonal mutations of NOTCH1, BIRC3, SF3B1 and TP53 were found, including ATM and other potential progression driving mutations. CNA analysis identified 31 lesions, none with known poor prognostic impact. No novel recurrent lesion was identified: most cases showed no lesions (38%) or an isolated del(13q) (31%). The expression of 6 genes, selected from a gene expression profile analysis by microarray and quantified by droplet digital PCR on a cohort of 79 CLL (58 US-CLL and 21 non-US-CLL), allowed to build a decision-tree capable of recognizing at diagnosis US-CLL patients. Conclusions: The genetic landscape of US-CLL is characterized by the absence of known unfavorable driver mutations/ CNA and of novel recurrent genetic lesions. Among CLL patients with favorable immunogenetics, a decision-tree based on the expression of 6 genes may identify at diagnosis patients who are likely to maintain an indolent disease for decades.
the pathways of NK cell/ALL blast interaction still need to be better clarified. In this study, w... more the pathways of NK cell/ALL blast interaction still need to be better clarified. In this study, we addressed the pathways of NK cell/ALL blast recognition and compared the expression of activating ligands among pediatric and adult ALL patients. Molecularly-defined subgroups of ALL patients were considered in the analysis, and functional tests were performed to confirm the results of the phenotypic findings. Methods Patients A total of 103 newly diagnosed cases of ALL, including 46 adults (median age 34 years; range 18-74 years) and 57 children (median age 4 years; range 0.1-17 years) were investigated between December 2011 and February 2013. Based on the immunophenotypic profile, the case series was subdivided as follows: 90 B-lineage ALL (B-ALL) (39 adult and 51 pediatric cases) and 13 T-lineage ALL (T-ALL) (7 adult and 6 pediatric cases). Within the adult BALL cohort, the following fusion transcripts were identified: BCR-ABL in 15 cases, MLL-AF4 in 7, E2A-PBX1 in 2, while the remaining 15 cases were negative for the most common molecular lesions found in ALL. In pediatric BALL , the BCR-ABL transcript was found in 6 patients, MLL-AF4 in 3, MLL-ENL in 1, TEL-AML1 in 10; 31 patients did not show any of the molecular aberrations investigated. Informed consent for biological studies was obtained from patients or their legal guardians in accordance with the Declaration of Helsinki. The study was approved by the local ethics committee.
Chronic lymphocytic leukemia (CLL) presents a highly variable clinical course, with some patients... more Chronic lymphocytic leukemia (CLL) presents a highly variable clinical course, with some patients surviving for many years without requiring treatment, while others have rapidly progressing disease, require aggressive treatment and show a short life expectancy. Several biological features of the leukemic cells - the mutational status of the immunoglobulin (Ig) heavy (H) chain variable (V) genes, cytogenetic alterations, ZAP-70 and CD38 antigen expression, p53 dysfunctions – bear prognostic value and allow to stratify patients into risk categories. The deletion of the chromosome region 11q22–q23 that occurs in 10–20% of cases represents the second most common genetic abnormality in CLL and defines a subgroup of patients characterized by advanced progressive disease and poor survival. The ATM (ataxia telangiectasia-mutated) gene is located in this region and several studies suggest that the 11q deletion results in ATM gene inactivation. The ATM gene consists of 66 exons, 62 (5–66) of which are coding exons. The ATM protein is a nuclear serine/threonine kinase of 350 kDa induced by chromosomal double-strand breaks that arise endogenously or after exposure to DNA-damaging agents, including ionizing radiations and drugs. It protects the integrity of the genome in several ways: it regulates the arrest of the cell cycle at the G1/S and G2/M phases to prevent the processing of damaged DNA; it activates DNA repair pathways and induces apoptosis if the DNA damage cannot be repaired. In the present study we examined the status of the ATM gene by DHPLC (Denaturing High Performance Liquid Cromatography) in a series of untreated CLL patients studied at the Division of Hematology, “Sapienza” University of Rome. In addition, a multiplex gene dosage analysis of the ATM gene was performed by MLPA (Multiplex Ligation Probe Amplification) in all patients. The results obtained have been correlated with the molecular status of the IgVH genes, the presence of cytogenetic abnormalities, ZAP-70 and CD38 expression, p53 mutations, and with the gene expression profile. Mutational screening of the ATM gene identified mutations in 8 of the 57 CLL patients studied (14%): 6 missense, 8095C>T (Pro2699Ser), 8071C>T (Arg2691Cys), 2476A>C (Ile826Leu) and, in 3 patients, 1435G>T (Asp479Tyr); 1 frameshift: 2502insA; 1 splicing mutation: IVS29+5G>A. In addition, 14 different variants and polymorphisms of unknown functional significance were found. The 8 ATM mutated cases showed the following features: 6 were IgVH unmutated, 4 were ZAP70+, 5 were CD38+, 4 harbored a 13q14 deletion, only 1 patient showed a 17p13 deletion and 2 a 14q32 deletion; furthermore, 2/8 patients showed a p53 gene mutation; none showed the 11q deletion. MLPA analysis identified 6 patients with 11q deletion and all of them showed at least 20% of cells positive for the 11q23 deletion evaluated by FISH; only 1 case showed a 17p13 deletion. In these cases, the IgVH gene was always unmutated and ZAP70 was positive; 4 cases were also CD38+. ATM gene mutated and deleted CLL cases showed a peculiar gene profile characterized by the up-regulation and down-regulation of genes involved in apoptosis and DNA repair function. On the contrary, no gene profile difference was found in patients carrying ATM gene polymorphisms. Patients with ATM gene mutated and deleted showed a short time to treatment, 23 and 19 months respectively. The results of this study indicate that ATM gene alterations - mutations or deletions - account for 24% of untreated CLL patients, are associated with a defined gene profile and are characterized by a poor prognosis. It is important to underline that these patients have been evaluated before any treatment and that ATM mutations have been found in the absence of 11q23 deletions. ATM alterations appear to represent the most frequent genetic abnormality with unfavorable prognostic implications in CLL and should be investigated prior to initiating therapy in order to plan an appropriate treatment algorithm.
CD4+CD25+ T-regulatory (Treg) cells have been shown to regulate self and allograft tolerance and ... more CD4+CD25+ T-regulatory (Treg) cells have been shown to regulate self and allograft tolerance and to suppress allogeneic immune response. Studies in mouse models of bone marrow (BM) transplantation have shown that the infusion of culture-expanded Tregs can be effective in preventing and suppressing graft-versus-host disease (GVHD), while still allowing a graft-versus-leukemia effect (GVL). Cord blood (CB) represents the most recent source of hematopietic stem cell transplantation (HSCT) used in the unrelated transplant setting. Comparative studies have shown that unrelated CB transplant is an acceptable alternative to unrelated BM transplantation, characterized by a lower risk of transplant-related mortality due to GVHD. CB has been reported to contain Tregs, but minimal data are available on these cells. Aim of this study was to compare the suppressive functions of Tregs obtained from CB units with those obtained from the peripheral blood (PB) of patients who have undergone an allogeneic BM transplantation and of normal donors. Treg cells were purified from mononuclear cells obtained from CB units or PB using the CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and expanded for 6 days in 96-well U-Bottom plates coated with anti-CD3 and anti-CD28 MoAbs plus IL-2. To assess the suppressive functions of Treg cells, expanded CD4+CD25+ Tregs from CB units or PB were seeded with naïve autologous effector CD4+CD25- T cells stimulated with allogeneic dendritic cells (DC) pulsed with apoptotic leukemic blasts, then incubated with [3H]-thymidine and counted in a beta-counter. Suppressor activity was measured as [3H]-thymidine incorporation in the presence or absence of Tregs. The immunophenotypic analysis of Tregs from CB units - in terms of expression of surface CD4, CD25, CD62L, cytoplasmic CTLA-4 and Foxp3 - before and after expansion, did not show any specific peculiarity. CB units (n = 6) contained a proportion of Tregs comparable to those contained in the PB of allotransplanted patients (n = 8) or in the PB of normal donors (n = 8) (mean % ± SD, CB 0.28% ± 0.41; PB patients 0.4% ± 0.4; PB donors CB 0.45% ± 0.29). On the contrary, Tregs from CB units and from the PB of allotransplanted patients showed a higher expansion capacity when compared to Tregs from the PB of normal donors (mean fold increase ± SD, CB units 11.5 ± 9.0; PB patients 9.6 ± 5.0; PB donors CB 5.2 ± 2.7). Preliminary data also show that Tregs expanded from CB units (n = 2) exert a higher suppressive function on the proliferative reaction of T cells stimulated by allogeneic DC if compared to Tregs expanded from the PB of allotransplanted patients (n = 2) (mean fold reduction ± SD, CB 5.25 ± 3.7; PB patients 3.8 ± 3.3). These results demonstrate that Treg cells contained in CB units present an expansion capacity superior to that of Tregs from PB of normal donors; moreover, these expanded cells seem to exert a potent suppressive function of the proliferative effect in mixed lymphocyte reaction culture assays. These data offer further insights in the understanding of the biology of CB transplantation and may indicate a possible clinical use of expanded Tregs for the prevention and management of GVHD in the setting of CB transplantation.
The capacity to generate effective dendritic cells (DC) from adult acute lymphoblastic leukemia (... more The capacity to generate effective dendritic cells (DC) from adult acute lymphoblastic leukemia (ALL) patients in complete remission (CR) and off-therapy was investigated. Monocyte-derived DC cultured in the presence of granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4 and tumor necrosis factor (TNF)-alpha expressed maturation markers, produced IL-12 and loaded apoptotic bodies to a similar extent to normal DC. Patients' circulating T and NK lymphocytes were normally represented and, after stimulation, were capable of producing TNF-alpha and interferon-gamma to a similar extent to control lymphocytes. DC loaded with leukemia-derived apoptotic bodies increased their ability to stimulate both allogeneic and autologous lymphocytes, and to generate specific anti-leukemic CD3 + cells. These findings offer a rationale for the design of DC-based vaccine programs for adult ALL patients in CR with the aim of controlling/eradicating the disease.
The immunologic reconstitution is ultimately responsible of the clinical outcome of patients who ... more The immunologic reconstitution is ultimately responsible of the clinical outcome of patients who have undergone an allogeneic stem cell transplantation (SCT). The occurrence of graft-versus-host disease (GVHD), which represents the major cause of morbidity and mortality after the transplant correlates with the concentration in the peripheral blood (PB) of regulatory T cells (Tregs). In this study we aim at demonstrating that not only the concentration but also the functional capacities and the degree of activity of Tregs act as an important regulator of alloreactivity and may help to predict the risk of acute and chronic GVHD in the post-transplant period. Sixteen patients who underwent an allogeneic SCT were evaluated at 1 year from transplant. Tregs were expanded from the PB of these patients and from 8 normal donors; their expansion capacity, phenotype, suppressor activity and IL-10 production were measured. Tregs expanded from patients without GVHD exerted a higher suppressive function on the proliferative reaction of T cells and showed a higher IL-10 production capacity compared to patients with acute or chronic GVHD. These results document that the functional activity and the suppressor capacity of Tregs after an allogeneic SCT may protect from GVHD, and support the design of clinical protocols based on the infusion of expanded and activated Tregs.
Background: CLL is characterized by an heterogeneous clinical course, with some patients living m... more Background: CLL is characterized by an heterogeneous clinical course, with some patients living many years without therapy and other patients presenting an aggressive disease. Different biologic properties of the leukemic cells have important prognostic implications. In particular, the IgVH gene mutational status, chromosomal abnormalities, expression of CD38 and ZAP-70 have been correlated with the clinical course of the disease. There is also growing evidence that angiogenesis may be linked to the pathogenesis and outcome of CLL. In particular, vascular endothelial growth factor A (VEGF-A) has been detected in the serum and bone marrow biopsy cells analyzed by immunostaining: both events have been correlated with a poor prognostic likelihood. Aims: The goal of this study was to obtain a more complete characterization of the angiogenic potential of CLL cells, to correlate the angiogenic capacity with other prognostic parameters and to investigate the possibility of overcoming this property by specific VEGF-signaling inhibitors. Methods: Eighteen patients with a diagnosis of CLL, based on morphologic and immunophenotypic criteria, have been evaluated for the expression of CD38 and ZAP-70, and for the IgVH gene mutational status. An Angiokit test (TCS Cell Works) was utilized to evaluate the capacity of purified CD19+ CLL cells to stimulate the growth and tubule formation of human endothelial cells and the relative roles of an anti-VEGF specific antibody and of PTK787/ZK 222584 (PTK/ZK, Schering, Novartis), a novel, oral angiogenesis and lymphangiogenesis inhibitor that blocks tyrosine kinase signaling from all known VEGFRs. The quantification of endothelial tubule formation was carried out using a specific software. After stimulation with a biotinylated goat anti-human IgM F(ab)2, leukemia cells were evaluated for gene expression profiling using the Affymetrix HGU133 Plus 2.0 gene chip. Results: In 9 patients, the leukemic cells were CD38+, ZAP-70+ and showed an unmutated IgVH status; in the remaining 9, the leukemic cells were CD38−, ZAP-70− and with a mutated IgVH status. In CLL patients with poor prognostic features - CD38+, ZAP-70+ and unmutated IgVH - purified leukemic cells showed a significantly greater capacity to stimulate the growth and tubule formation (3407 ± 968 tubules) of human endothelial cells compared to leukemic cells obtained from CLL patients with good prognostic factors, i.e. CD38−, ZAP-70− and mutated IgVH, (1550 ± 624 tubules). This property was amplified following sIgM cross-linking, that resulted in the upregulation of angiogenetic genes, including: EGR-1, SDF2L1, TNFAIP3, PTGER4, FIBP, NR4A3. A reduction of VEGF-A-induced tubule number was observed when leukemic cells were cultured with the anti-VEGF antibody (43–82% of inhibition) and, to a further extent, with the PTK/ZK inhibitor (90–96% of inhibition) compared to purified CLL cells alone. Conclusions: In CLL patients with poor prognostic factors, leukemic cells display an angiogenic capability significantly higher than that of cells from patients with favorable prognostic factors. The current findings provide the rationale for investigating the potential clinical role of anti-angiogenic agents, such as PTK/ZK, in the management of CLL patients.
Uploads
Papers by Nadia Peragine