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Chronic Lymphocytic Leukemia (CLL) is an indolent malignancy characterized by the accumulation of quiescent mature B cells. However, these cells are transcriptionally and translationally active, implicating an active metabolism. The recent literature suggests that CLL cells have an oxidative-type phenotype. Given the role of cell metabolism, which is able to influence the outcome of treatments, in other neoplasms, we aimed to assess its prognostic role in CLL patients by determining the ex vivo bioenergetic metabolic profile of CLL cells, evaluating the correlation with the patient clinical/biological characteristics and the in vivo response to BTK inhibitor treatment. Clustering analysis of primary samples identified two groups, characterized by low (CLL low) or high (CLL high) bioenergetic metabolic rates. Compared to the CLL high, CLL with lower bioenergetic metabolic rates belonged to patients characterized by a statistically significant higher white blood cell count and by unfavorable molecular genetics. More importantly, patients in the CLL low cluster displayed a better and more durable response to the BTK inhibitor ibrutinib, thus defining a bioenergetic metabolic subgroup that can benefit the most from this therapy.

Chronic lymphocytic leukemia (CLL) is characterized by low CD20 expression, in part explained by an epigenetic-driven downregulation triggered by mutations of the NOTCH1 gene. In the present study, by taking advantage of a wide and well-characterized CLL cohort (n=537), we demonstrate that CD20 expression is downregulated in SF3B1-mutated CLL to an extent similar to NOTCH1-mutated CLL. In fact, SF3B1-mutated CLL cells show common features with NOTCH1- mutated CLL cells, including a gene expression profile enriched in NOTCH1-related gene sets and elevated expression of the active intracytoplasmic NOTCH1. Activation of the NOTCH1 signaling and downregulation of surface CD20 in SF3B1-mutated CLL cells correlate with overexpression of an alternatively spliced form of DVL2, a component of the Wnt pathway and negative regulator of the NOTCH1 pathway. These findings were confirmed by separately analyzing the CD20dim and CD20bright cell fractions from SF3B1-mutated cases as well as by DVL2 knockout experiments in CLL-like cell models. Together, the clinical and biological features that characterize NOTCH1-mutated CLL may also be recapitulated in SF3B1-mutated CLL, contributing to explain the poor prognosis of this CLL subset and providing the rationale for expanding therapies based on novel agents to SF3B1-mutated CLL.

The introduction of agents inhibiting the BCR-associated kinases such as ibrutinib has dramatically changed treatments algorithms of chronic lymphocytic leukaemia (CLL) as well as the role of different adverse prognosticators. We evaluated the efficacy of ibrutinib as single agent, in a real-life context, on 180 patients with CLL mostly pre-treated, recruited from three independent cohorts from Italy. Patients received 420 mg oral ibrutinib once daily until progression or occurrence of unacceptable side effects. Seventy-three patients discontinued ibrutinib for progression or for adverse events. NOTCH1 mutations (M) were correlated with a reduced redistribution lymphocytosis, calculated at 3 months on ibrutinib (p=0.022). Moreover, NOTCH1 mutated patients showed inferior nodal response at 6 months on ibrutinib compared to NOTCH1 wild type patients (p

We present a laboratory-based prognostic calculator (designated CRO score) to risk stratify treatment-free survival in early stage (Rai 0) chronic lymphocytic leukemia (CLL) developed using a training-validation model in a series of 1,879 cases from Italy, the United Kingdom and the United States. By means of regression analysis, we identified five prognostic variables with weighting as follows: deletion of the short arm of chromosome 17 and unmutated immunoglobulin heavy chain gene status, 2 points; deletion of the long arm of chromosome 11, trisomy of chromosome 12, and white blood cell count >32.0x103/microliter, 1 point. Low-, intermediate- and high-risk categories were established by recursive partitioning in a training cohort of 478 cases, and then validated in four independent cohorts of 144 / 395 / 540 / 322 cases, as well as in the composite validation cohort. Concordance indices were 0.75 in the training cohort and ranged from 0.63 to 0.74 in the four validation cohorts (0.69 in the composite validation cohort). These findings advocate potential application of our novel prognostic calculator to better stratify early-stage CLL, and aid case selection in risk-adapted treatment for early disease. Furthermore, they support immunocytogenetic analysis in Rai 0 CLL being performed at the time of diagnosis to aid prognosis and treatment, particularly in today’s chemofree era.

BIRC3 is a recurrently mutated gene in chronic lymphocytic leukemia (CLL) but the functional implications of BIRC3 mutations are largely unexplored. Furthermore, little is known about the prognostic impact of BIRC3 mutations in CLL cohorts homogeneously treated with first-line fludarabine, cyclophosphamide, and rituximab (FCR). By immunoblotting analysis, we showed that the non-canonical nuclear factor-κB pathway is active in BIRC3-mutated cell lines and in primary CLL samples, as documented by the stabilization of MAP3K14 and by the nuclear localization of p52. In addition, BIRC3-mutated primary CLL cells are less sensitive to flu-darabine. In order to confirm in patients that BIRC3 mutations confer resistance to fludarabine-based chemoimmunotherapy, a retrospective multicenter cohort of 287 untreated patients receiving first-line FCR was analyzed by targeted next-generation sequencing of 24 recurrently mutated genes in CLL. By univariate analysis adjusted for multiple comparisons BIRC3 mutations identify a poor prognostic subgroup of patients in whom FCR treatment fails (median progression-free survival: 2.2 years, P

Abstract Background The stromal vascular fraction (SVF) derived from adipose tissue contains adipose-derived stromal/stem cells (ASC) and can be used for regenerative applications. Thus, a validated protocol for SVF isolation, freezing, and thawing is required to manage product administration. To comply with Good Manufacturing Practice (GMP), fetal bovine serum (FBS), used to expand ASC in vitro, could be replaced by growth factors from platelet concentrates. Methods Throughout each protocol, GMP-compliant reagents and devices were used. SVF cells were isolated from lipoaspirates by a standardized enzymatic protocol. Cells were cryopreserved in solutions containing different albumin or serum and dimethylsulfoxide (DMSO) concentrations. Before and after cryopreservation, we analyzed: cell viability (by Trypan blue); immunophenotype (by flow cytometry); colony-forming unit-fibroblast (CFU-F) formation; and differentiation potential. ASC, seeded at different densities, were expanded in presence of 10% FBS or 5% supernatant rich in growth factors (SRGF) from platelets. The differentiation potential and cell transformation grade were tested in expanded ASC. Results We demonstrated that SVF can be obtained with a consistent yield (about 185 × 103 cells/ml lipoaspirate) and viability (about 82%). Lipoaspirate manipulation after overnight storage at +4 °C reduced cell viability (−11.6%). The relative abundance of ASC (CD34+CD45−CD31–) and endothelial precursors (CD34+CD45−CD31+) in the SVF product was about 59% and 42%, respectively. A period of 2 months cryostorage in autologous serum with added DMSO minimally affected post-thaw SVF cell viability as well as clonogenic and differentiation potentials. Viability was negatively affected when SVF was frozen at a cell concentration below 1.3 × 106 cells/ml. Cell viability was not significantly affected after a freezing period of 1 year. Independent of seeding density, ASC cultured in 5% SRGF exhibited higher growth rates when compared with 10% FBS. ASC expanded in both media showed unaltered identity (by flow cytometry) and were exempt from genetic lesions. Both 5% SRGF- and 10% FBS-expanded ASC efficiently differentiated to adipocytes, osteocytes, and chondrocytes. Conclusions This paper reports a GMP-compliant approach for freezing SVF cells isolated from adipose tissue by a standardized protocol. Moreover, an ASC expansion method in controlled culture conditions and without involvement of animal-derived additives was reported.

Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous disease characterised 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. Stimulation through the B cell receptor (BCR) plays a prominent role in the selection and expansion of the malignant clone in CLL. On the other hand, other external signals delivered by several cell types including T lymphocytes, macrophages, stromal cells, endothelial cells, and follicular dendritic cells, operating through either direct BCR-independent cell-cell contact or indirect production of paracrine soluble factors, synergistically cooperate in regulating proliferation and survival of CLL cells. In this context, CD49d is known to play a pivotal role in mediating both cell-cell and cell-matrix interactions in CLL-involved tissues, eventually delivering pro-survival signals and protecting CLL cells from drug-induced damages. In the present review, we focused on functional and physical interactions of CD49d with other microenvironmental receptors, including CD38 and BCR, and other specific CD49d-dependent interactions in lymph node and bone marrow microenvironments responsible for growth and survival-supporting signals, eventually influencing CLL prognosis and therapeutic options.

In chronic lymphocytic leukemia the balance between the pro-apoptotic and anti-apoptotic members of the bcl-2 family is involved in the pathogenesis, chemorefractoriness and clinical outcome. Moreover, the recently proposed anti-bcl-2 molecules, such as ABT-199, have emphasized the potential role of of bcl-2 family proteins in the context of target therapies. We investigated bax/bcl-2 ratio by flow cytometry in 502 patients and identified a cut off of 1.50 to correlate bax/bcl-2 ratio with well-established clinical and biological prognosticators. Bax/bcl-2 was 1.50 or over in 263 patients (52%) with chronic lymphocytic leukemia. Higher bax/bcl-2 was associated with low Rai stage, lymphocyte doubling time over 12 months, beta-2 microglobulin less than 2.2 mg/dL, soluble CD23 less than 70 U/mL and a low risk cytogenetic profile (P

Background CD69 is expressed in several hemopoietic cells and is an early activation marker in chronic lymphocytic leukemia. Chronic lymphocytic leukemia is a clinically heterogeneous disease which needs novel prognostic parameters which can be easily and efficiently managed.Design and Methods We investigated CD69 by flow cytometry in a series of 417 patients affected by chronic lymphocytic leukemia and compared this to other biological and clinical prognosticators.Results CD69 was associated with Rai stages (P=0.00002), β2-microglobulin (P=0.0005) and soluble CD23 (P

Identification of prognosticators for Binet A chronic lymphocytic leukemia is important for selecting patients with dismal prognosis. We analyzed CD49d expression in 140 consecutive Binet A chronic lymphocytic leukemia. At diagnosis, CD49d ≥30% (54/140, 38.6%) associated with proliferation markers, namely CD38 ≥30% (p=3.9×10−6), LDH (p=0.007) and β2-microglobulin (p=0.020). Univariate log-rank analysis identified CD49d ≥30% as a risk factor of treatment free survival (p=8.3x10−5), time to progression to a more advanced stage (p=4.7×10−4), and time to lymphocyte doubling (p=0.009). Multivariate analysis selected CD49d ≥30% as an independent treatment free survival predictor after adjustment for biological (HR 2.28; 95% CI 1.71–4.45, p=0.015) and both biological and clinical variables analyzed together (HR 3.33, 95% CI 1.61–6.90, p=0.001). Within Binet A subgroups harboring favorable biological variables (IGHV homology