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Chronic lymphocytic leukaemia is the most prevalent leukaemia in Western countries. It is an incurable disease characterized by a highly variable clinical course. Chronic lymphocytic leukaemia is an ideal model for studying clonal heterogeneity and dynamics during cancer progression, response to therapy and/or relapse because the disease usually develops over several years. Here we report an analysis by deep sequencing of sequential samples taken at different times from the affected organs of two patients with 12- and 7-year disease courses, respectively. One of the patients followed a linear pattern of clonal evolution, acquiring and selecting new mutations in response to salvage therapy and/or allogeneic transplantation, while the other suffered loss of cellular tumoral clones during progression and histological transformation.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease without a well-defined genetic alteration responsible for the onset of the disease. Several lines of evidence coincide in identifying stimulatory and growth signals delivered by B-cell receptor (BCR), and co-receptors together with NFkB pathway, as being the driving force in B-cell survival in CLL. However, the molecular mechanism responsible for this activation has not been identified. Based on the hypothesis that BCR activation may depend on somatic mutations of the BCR and related pathways we have performed a complete mutational screening of 301 selected genes associated with BCR signaling and related pathways using massive parallel sequencing technology in 10 CLL cases. Four mutated genes in coding regions (KRAS, SMARCA2, NFKBIE and PRKD3) have been confirmed by capillary sequencing. In conclusion, this study identifies new genes mutated in CLL, all of them in cases with progressive disease, and demonstrates that next-generation sequencing technologies applied to selected genes or pathways of interest are powerful tools for identifying novel mutational changes.

Matrix metalloproteinase-9 (MMP-9) contributes to chronic lymphocytic leukemia (CLL) pathology by regulating cell migration and preventing spontaneous apoptosis. It is not known if MMP-9 is involved in CLL cell response to chemotherapy and we address this in the present study, using arsenic trioxide (ATO) and fludarabine as examples of cytotoxic drugs. We used primary cells from the peripheral blood of CLL patients and MEC-1 cells stably transfected with an empty vector or a vector containing MMP-9. The effect of ATO and fludarabine was determined by flow cytometry and by the MTT assay. Expression of mRNA was measured by RT-PCR and qPCR. Secreted and cell-bound MMP-9 was analyzed by gelatin zymography and flow cytometry, respectively. Protein expression was analyzed by Western blotting and immunoprecipitation. Statistical analyses were performed using the two-tailed Student's t-test. In response to ATO or fludarabine, CLL cells transcriptionally upregulated MMP-9, preceding the onset of apoptosis. Upregulated MMP-9 primarily localized to the membrane of early apoptotic cells and blocking apoptosis with Z-VAD prevented MMP-9 upregulation, thus linking MMP-9 to the apoptotic process. Culturing CLL cells on MMP-9 or stromal cells induced drug resistance, which was overcome by anti-MMP-9 antibodies. Accordingly, MMP-9-MEC-1 transfectants showed higher viability upon drug treatment than Mock-MEC-1 cells, and this effect was blocked by silencing MMP-9 with specific siRNAs. Following drug exposure, expression of anti-apoptotic proteins (Mcl-1, Bcl-xL, Bcl-2) and the Mcl-1/Bim, Mcl-1/Noxa, Bcl-2/Bax ratios were higher in MMP-9-cells than in Mock-cells. Similar results were obtained upon culturing primary CLL cells on MMP-9. Our study describes for the first time that MMP-9 induces drug resistance by modulating proteins of the Bcl-2 family and upregulating the corresponding anti-apoptotic/pro-apoptotic ratios. This is a novel role for MMP-9 contributing to CLL progression. Targeting MMP-9 in combined therapies may thus improve CLL response to treatment.

We previously showed that MMP-9 contributes to CLL pathology by regulating cell survival and migration and that, when present at high levels, MMP-9 induces cell arrest. To further explore the latter function, we studied whether MMP-9 influences the gene-expression profile in CLL. Microarray analyses rendered 131 differentially expressed genes in MEC-1 cells stably transfected with MMP-9 (MMP-9-cells) versus cells transfected with empty vector (Mock-cells). Ten out of twelve selected genes were also differentially expressed in MEC-1 cells expressing the catalytically inactive MMP-9MutE mutant (MMP-9MutE-cells). Incubation of primary CLL cells with MMP-9 or MMP-9MutE also regulated gene and protein expression, including CD99, CD226, CD52, and CD274. Because CD99 is involved in leukocyte transendothelial migration, we selected CD99 for functional and mechanistic studies. The link between MMP-9 and CD99 was reinforced with MMP-9 gene silencing studies, which resulted in CD99 upregulation. CD99 gene silencing significantly reduced CLL cell adhesion, chemotaxis and transendothelial migration, while CD99 overexpression increased cell migration. Mechanistic analyses indicated that MMP-9 downregulated CD99 via binding to α4β1 integrin and subsequent inactivation of the Sp1 transcription factor. This MMP-9-induced mechanism is active in CLL lymphoid tissues, since CD99 expression and Sp1 phosphorylation was lower in bone marrow-derived CLL cells than in their peripheral blood counterparts. Our study establishes a new gene regulatory function for MMP-9 in CLL. It also identifies CD99 as an MMP-9 target and a novel contributor to CLL cell adhesion, migration and arrest. CD99 thus constitutes a new therapeutic target in CLL, complementary to MMP-9.

Chronic Lymphocytic Leukemia (CLL) is the most prevalent leukemia in Western countries and is notable for its variable clinical course. This variability is partly reflected by the mutational status of IGHV genes. Many CLL samples have been studied in recent years by next-generation sequencing. These studies have identified recurrent somatic mutations in NOTCH1 , SF3B1 , ATM , TP53 , BIRC3 and others genes that play roles in cell cycle, DNA repair, RNA metabolism and splicing. In this study, we have taken a deep-targeted massive sequencing approach to analyze the impact of mutations in the most frequently mutated genes in patients with CLL enrolled in the REM ( rituximab en mantenimiento ) clinical trial. The mutational status of our patients with CLL, except for the TP53 gene, does not seem to affect the good results obtained with maintenance therapy with rituximab after front-line FCR treatment.

Mutational status of together with expression of wild-type (wt) represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on and mutational status. Cells from patients with worse prognosis (mutated and wt ) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.

The aim of this study was to assess the incidence of cytomegalovirus (CMV) infection and disease in patients with hematologic malignancies treated with alemtuzumab. The outcome of CMV infection in hematologic patients treated with alemtuzumab in 19 hospitals throughout Spain was assessed retrospectively. Data were collected from the medical records of patients over a period of 6 months following initiation of alemtuzumab therapy. We studied 102 patients (89 with B-cell chronic lymphocytic leukemia and 13 with other lymphoproliferative diseases, with a median age of 63 years [range 29-81 years]). Alemtuzumab was administered for a mean of 11.2 (standard deviation: 13.8) weeks, with a median total dose of 423 mg (range: 59-1440 mg). Alemtuzumab as a single agent was administered in 92.2% of patients and was associated with chemotherapy in 7.8% of cases. Prophylactic antivirals included famcyclovir (47%), acyclovir (34%), valacyclovir (14%) and valgancyclovir (5%). CMV viremia testing was performed a mean of 6.3 times (range: 1-19). The incidence of CMV infection was 38.9% (46% in patients treated with steroids and 75% in patients receiving ≥1000 mg of alemtuzumab). Treatment of CMV infection included gancyclovir or valgancyclovir in 94% of cases. Viremia became negative after a median of 20 days (95% CI: 13.4-26.6). CMV disease occurred in five patients. The incidence of CMV infection in alemtuzumab-treated patients was 38.9%. The incidence increased in patients treated concomitantly with steroids and in those treated with high doses of alemtuzumab, although only eight patients received 1000 mg or more, systematic monitoring of CMV viremia and early treatment of infection resulted in a favorable outcome of CMV reactivation.

Diffuse large B-cell lymphoma (DLBCL) requires a complete staging at diagnosis that may have prognostic and therapeutic implications. The role of bone marrow (BM) biopsy (BMB) is controversial in the era of nuclear imaging techniques. We performed a comparative review of 25 studies focused on BM evaluation at DLBCL diagnosis, including at least two of the following techniques: BMB, flow cytometry, and positron emission tomography (PET-FDG). The report about BM involvement (BMi), diagnostic accuracy, and prognostic significance was collected and compared among techniques. A concordance analysis between BMB, FCM, and PET was also performed, and we deeply evaluated the implications of the different types of BMi: concordant by LBCL or discordant by low-grade B-cell lymphoma for both BMB and FCM, and focal or diffuse uptake pattern for PET. As a main conclusion, BMB, FCM, and PET are complementary tools that provide different and clinically relevant information in the assessment of BMi in newly diagnosed DLBCL.

In chronic lymphocytic leukemia, the reliability of next‐generation sequencing (NGS) to detect TP53 variants ≤10% allelic frequency (low‐VAF) is debated. We tested the ability to detect 23 such variants in 41 different laboratories using their NGS method of choice. The sensitivity was 85.6%, 94.5%, and 94.8% at 1%, 2%, and 3% VAF cut‐off, respectively. While only one false positive (FP) result was reported at >2% VAF, it was more challenging to distinguish true variants <2% VAF from background noise (37 FPs reported by 9 laboratories). The impact of low‐VAF variants on time‐to‐second‐treatment (TTST) and overall survival (OS) was investigated in a series of 1092 patients. Among patients not treated with targeted agents, patients with low‐VAF TP53 variants had shorter TTST and OS versus wt‐TP53 patients, and the relative risk of second‐line treatment or death increased continuously with increasing VAF. Targeted therapy in ≥2 line diminished the difference in OS between patients with low‐VAF TP53 variants and wt‐TP53 patients, while patients with high‐VAF TP53 variants had inferior OS compared to wild type‐TP53 cases. Altogether, NGS‐based approaches are technically capable of detecting low‐VAF variants. No strict threshold can be suggested from a technical standpoint, laboratories reporting TP53 mutations should participate in a standardized validation set‐up. Finally, whereas low‐VAF variants affected outcomes in patients receiving chemoimmunotherapy, their impact on those treated with novel therapies remains undetermined. Our results pave the way for the harmonized and accurate TP53 assessment, which is indispensable for elucidating the role of TP53 mutations in targeted treatment.

The assembly of a collection of gene-expression signatures of the major types of B-cell non-Hodgkin's lymphoma has identified increased T-cell leukemia/lymphoma 1A (TCL1) expression in multiple lymphoma types and cases, and has enabled the investigation of the functional and clinical importance of TCL1 expression. Specifically, Burkitt's lymphoma cases show a homogeneously strong expression of TCL1, whereas diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, nodal marginal zone lymphoma, and splenic marginal zone lymphoma display a striking variability in the intensity of TCL1 staining. This was validated in two independent series. A Gene-Set Enrichment Analysis of the genes correlated with TCL1A expression found that variation in the level of expression of TCL1A was significantly associated with some of the most important gene signatures recognizing B-cell lymphoma pathogenesis and heterogeneity, such as germinal center, B-cell receptor, NF-κB (and its target genes), death, MAP kinases, TNFR1, TOLL, and IL1R. Additionally, TCL1 expression was correlated with shorter time to treatment in chronic lymphocytic leukemia cases and shorter lymphoma-specific survival in mantle cell lymphoma series, thus indicating the clinical and biological significance of TCL1 expression, and suggesting TCL1A as a potential therapeutic target.