Search

To assess the involvement of microRNAs (miRNAs) in B-cell receptor (BCR) stimulation, we first evaluated miRNA profiling following IgM cross-linking in chronic lymphocytic leukemia (CLL) cells and in normal B lymphocytes. Second, we combined miRNA and gene expression data to identify putative miRNA functional networks. miRNA profiling showed distinctive patterns of regulation after stimulation in leukemic versus normal B lymphocytes and identified a differential responsiveness to BCR engagement in CLL subgroups according to the immunoglobulin heavy chain variable region mutational status and clinical outcome. The most significantly modulated miRNAs in stimulated CLL are miR-132 and miR-212. Notably, these miRNAs appeared regulated in progressive but not in stable CLL. Accordingly, gene profiling showed a significant transcriptional response to stimulation exclusively in progressive CLL. Based on these findings, we combined miRNA and gene expression data to investigate miR-132 and miR-212 candidate interactions in this CLL subgroup. Correlation analysis pointed to a link between these miRNAs and RB/E2F and TP53 cascades with proproliferative effects, as corroborated by functional analyses. Finally, basal levels of miR-132 and miR-212 were measured in an independent cohort of 20 unstimulated CLL cases and both showed lower expression in progressive compared to stable patients, suggesting an association between the expression of these molecules and disease prognosis. Overall, our results support a model involving miR-132 and miR-212 upregulation in sustaining disease progression in CLL. These miRNAs may therefore provide new valuable strategies for therapeutic intervention. © 2015 Wiley Periodicals, Inc.

In chronic lymphocytic leukemia, NOTCH1, SF3B1, BIRC3 and TP53 disruptions are recurrent and affect survival. To define their incidence and clinical impact in patients undergoing first-line treatment, we evaluated 163 cases enrolled in the GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) LLC0405 protocol (fludarabine plus alemtuzumab or fludarabine plus cyclophosphamide), for young patients, or in the ML21445 protocol (chlorambucil plus rituximab), for elderly patients. NOTCH1, SF3B1, BIRC3 and TP53 disruptions were detected in 15.9%, 12.2%, 8.6% and 10.4% of cases. NOTCH1 mutations correlated with a shorter treatment-free interval (p = 0.058), an unmutated immunoglobulin heavy variable gene (IGHV) status (p0.0001), CD38 and ZAP-70 expression (p = 0.0025 and 0.026, respectively) and trisomy 12 (p = 0.0028), SF3B1 mutations with an unmutated IGHV status (p = 0.02), and BIRC3 disruptions with an unmutated IGHV configuration (p = 0.01) and 11q deletion (p0.0001). NOTCH1 and SF3B1 did not appear to impact on overall response, while an inferior response was observed for BIRC3- and TP53-disrupted cases in the LLC0405 and ML21445 protocols, respectively. Progression-free survival, evaluable in the LLC0405 protocol - not affected by NOTCH1, SF3B1 and TP53 - appeared inferior for BIRC3 disruption. NOTCH1 and SF3B1 mutations may be overcome by aggressive regimens, while BIRC3 might impact on outcome also in intensive regimens.

Background and aims. B-lineage acute lymphoblastic leukemia (B-ALL) carrying t(4;11)(q21;q23)/KMT2A-AFF1 accounts for roughly 10% of adult B-ALL, it is associated to pro-B immunophenotype and aggressive clinical features. However, the role of KMT2A-AFF1 in pediatric-like, minimal residual disease (MRD)-driven clinical trials and the impact of transplant in this poor prognostic subgroup are still debated. To address these issues we investigated the incidence, clinical features and outcome of KMT2A-AFF1-positive ALL treated in 6 consecutive GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) clinical trials. Materials and methods. Between November 1996 and September 2016, 926 BCR-ABL1-negative B-ALL patients were enrolled in the GIMEMA clinical trials LAL0496, LAL2000, LAL0904, LAL1104, LAL1308, LAL1913. The median follow-up is 25.4 months (range: 0.1-146.9). Starting from LAL2000, KMT2A-AFF1-positive patients were classified as high-risk and managed more intensively. LAL1308 and LAL1913 adopted a similar pediatric-like backbone, at variance from LAL0496, LAL2000 and LAL0904. Overall, median age was 34.3 (range, 14.1 - 81.9), there were 502 males (54.2%) and 424 females (45.8%) and the median WBC count was 8.80 x 109/L (range, 0.4- 872.3). Incidence, clinical features and outcome of KMT2A-AFF1-positive were analyzed in comparison with KMT2A-AFF1-negative B-ALL. MRD evaluation after induction treatment was available in 197 patients enrolled in the more recent protocols. Results. Overall, 97/926 (10.5%) samples harbored KMT2A-AFF1 fusion gene. The comparison between KMT2A-AFF1-positive and the 829 KMT2A-AFF1-negative B-ALL patients revealed that KMT2A-AFF1-positive patients had a higher median age (42 vs 32.5 years old, p Conclusions. The present study examined a cohort of 926 BCR-ABL1-negative B-ALL treated in Italian multicenter clinical trials. Overall, though not differing in the achievement of CR and MRD negativity, the analysis of survival curves confirms that KMT2A-AFF1-positive patients have a significantly shorter survival than KMT2A-AFF1-negative patients. This result holds true also when censoring for transplant, thus suggesting that allograft does not confer a survival advantage to KMT2A-AFF1-positive patients. Moreover, KMT2A-AFF1-positive patients perform poorly also in more intensive regimen, as emerged from the analysis of the GIMEMA LAL1913 clinical trial. These findings encourage the adoption of alternative strategies: among the available targeted therapies, preclinical models of KMT2A-leukemia show sensitivity to BCL2-inhibitors, thus providing the rationale for testing this approach in upcoming clinical trials for KMT2A-AFF1-positive patients. Figure Disclosures Chiaretti: Amgen: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Krampera:Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Di Raimondo:Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy. Bassan:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Vignetti:Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: Educational Training; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Educational Training.

Background BCR/ABL1-like (alias Ph-like) acute lymphoblastic leukemia (ALL) is a clinically challenging subgroup. Its incidence varies from 10% in children to 30% in adult ALL. The underlying genomic background, usually characterized by the presence of kinase activating lesions, opens the way to targeted treatment. However, one of the major issues of this subset is represented by the identification of a standardized assay for their recognition at diagnosis. Indeed, while gene expression profile (GEP) and next-generation sequencing (NGS) are efficient techniques to identify these cases, they are difficult to be implemented in the diagnostic routine. Our group previously developed a rapid, simple and cost-effective algorithm based on a quantitative real time-polymerase chain reaction (Q-RT-PCR) named "BCR/ABL1-like predictor". Briefly, the expression levels of 10 genes - NUDT4, SEMA6A, ADGRE5, SOCS2, JCHAIN, CRLF2, TP53INP1, CD99, IFITM1 and IFITM2 - are used to generate a score: cases with a score ≥-0.3 are classified as BCR/ABL1-like. Aims After developing the BCR/ABL1-like predictor, we sought to: further refine the ability of this model to correctly predict BCR/ABL1-like ALL by evaluating NGS and RNA-sequencing of BCR/ABL1-like and non-BCR/ABL1-like cases, and to define the incidence of specific lesions;evaluate the reproducibility of the predictor when performed in external laboratories or when samples with a known genetic background were analyzed in house by the predictor. Results NGS and RNA-sequencing were carried out in 28 BCR/ABL1-like cases: CRLF2 overexpression, was found 35.7% of cases, with 3 harboring a CRLF2 rearrangement and 1 with a concomitant CRLF2 mutation. Furthermore, 13 JAK/STAT pathway mutations - JAK1, n=5; JAK2, n=3; IL7R and CRLF2, n=2 each, JAK3, n=1 - were identified in 33.3% of cases. Finally, RNA-sequencing and/or FISH experiments of the BCR/ABL1-like ALL cases revealed 11 lesions: 5 ABL-class fusion genes (3 NUP214/ABL1, 1 ZC3HAV1/ABL2 and 1 EBF1/PDGFRB), 2 BCR/JAK2, 3 CRLF2-r and 1 DDX3X/USP9X. In order to verify the reproducibility of the predictor, a collaboration was started across Europe with the Institute of Hematology and Blood Transfusion (ÚHKT, Prague), Josep Carreras Leukaemia Research Institute (Barcelona) and Munich Leukemia Laboratory (Munich), through the exchange of material and/or data. The first collaboration was carried out with the ÚHKT and Josep Carreras Leukaemia Research Institute laboratories: 11 cDNA samples (from 1 μg of total RNA), previously studied by our laboratory and classified as BCR/ABL1-like (n=5) and non-BCR/ABL1-like (n=6) ALL were shipped blindly and evaluated for the model in these laboratories. The technical set-up was sent to each laboratory, consisting of experimental procedures, PCR protocol and the threshold for Q-RT-PCR analysis. We received the raw data (i.e. 2^(-ΔCt) values) and uploaded them in the on-line BCR/ABL1-like predictor tool. We had 100% concordance with the ÚHKT and 81.8% with the Josep Carreras Leukaemia Research Institute. The main discrepancies were with 2/5 BCR/ABL1-like cases resulted as non-BCR/ABL1-like in the external laboratory: 1 was a p210 BCR/ABL1-positive case, used as control, which proved borderline, and the second was a BCR/ABL1-like case that, on the other hand, was not studied for the genetic features. A further collaboration was carried out with the MLL laboratory: we received 12 RNA samples - already analyzed by NGS and FISH - for evaluation by the BCR/ABL1-like predictor. The BCR-ABL1-like predictor classified 5 cases as BCR/ABL1-like and 7 as non-BCR-ABL1-like: all 5 BCR/ABL1-like carried a typical signature, consisting of a CRLF2 rearrangement plus JAK/STAT pathway mutations. Discordant cases were represented by 2 non-BCR-ABL1-like cases: one had a CRLF2 rearrangement plus JAK/STAT mutations and the other an ABL1 rearrangement. It is important to underline that the latter case had a borderline score (-0.482). Thus, the concordance rate was 83.3%. Conclusions This study shows that the BCR/ABL1-like predictor is a valid and reproducible tool across laboratories to identify rapidly these cases, with the goal of introducing genetic-driven therapeutic approaches upfront. One critical aspect is represented by borderline cases: in these patients, NGS experiments are required to define the underlying genomic lesion. Further investigations are currently underway. Disclosures Chiaretti: Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees. Machova:Novartis: Consultancy; Incyte: Consultancy; BMS: Consultancy, Research Funding. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Foà:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Speakers Bureau.

Chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptor (BCR) belonging to subset #1 (IGHV1-5-7/ IGKV1-39) display a poor outcome. To characterize their genetic and genomic features and BCR function, we selected 20 subset #1 CLL from a series of 579 cases. Subset #1 CLL, all showing unmutated IGHV, were associated with the presence of del(11q) (50%) in comparison with unmutated CLL, unmutated stereotyped CLL other than subset #1 and with cases using the same IGHV genes but a heterogeneous VH CDR3 (non-subset #1 CLL). There were no distinctive features regarding CD38, ZAP-70, and TP53 disruption. NOTCH1, SF3B1, and BIRC3 were mutated in 15%, 0%, and 5% of cases, respectively, while BIRC3 was deleted in 22% of cases. Microarray unsupervised analysis on 80 unmutated/mutated/stereotyped/non-stereotyped CLL showed a tight clustering of subset #1 cases. Their genomic signature exhibited several differentially expressed transcripts involved in BCR signal transduction, apoptosis regulation, cell proliferation, and oxidative processes, regardless of del(11q). Accordingly, BCR ligation with anti-IgM revealed a significant higher proliferation of subset #1 versus unmutated non-subset #1 CLL, both at baseline and after 24–48 hr stimulation. Subset #1 CLL represent a paradigmatic example of the direct link between BCR structure, function, and patients prognosis.

Nodal marginal zone lymphoma (NMZL) is a rare, indolent B-cell tumor that is distinguished from splenic marginal zone lymphoma (SMZL) by the different pattern of dissemination. NMZL still lacks distinct markers and remains orphan of specific cancer gene lesions. By combining whole-exome sequencing, targeted sequencing of tumor-related genes, whole-transcriptome sequencing, and high-resolution single nucleotide polymorphism array analysis, we aimed at disclosing the pathways that are molecularly deregulated in NMZL and we compare the molecular profile of NMZL with that of SMZL. These analyses identified a distinctive pattern of nonsilent somatic lesions in NMZL. In 35 NMZL patients, 41 genes were found recurrently affected in ≥3 (9%) cases, including highly prevalent molecular lesions of MLL2 (also known as KMT2D; 34%), PTPRD (20%), NOTCH2 (20%), and KLF2 (17%). Mutations of PTPRD, a receptor-type protein tyrosine phosphatase regulating cell growth, were enriched in NMZL across mature B-cell tumors, functionally caused the loss of the phosphatase activity of PTPRD, and were associated with cell-cycle transcriptional program deregulation and increased proliferation index in NMZL. Although NMZL shared with SMZL a common mutation profile, NMZL harbored PTPRD lesions that were otherwise absent in SMZL. Collectively, these findings provide new insights into the genetics of NMZL, identify PTPRD lesions as a novel marker for this lymphoma across mature B-cell tumors, and support the distinction of NMZL as an independent clinicopathologic entity within the current lymphoma classification.

Recurrent deletions of the long arm of chromosome 5 were detected in 23/200 cases of T-cell acute lymphoblastic leukemia. Genomic studies identified two types of deletions: interstitial and terminal. Interstitial 5q deletions, found in five cases, were present in both adults and children with a female predominance (chi-square, P=0.012). Interestingly, these cases resembled immature/early T-cell precursor acute lymphoblastic leukemia showing significant down-regulation of five out of the ten top differentially expressed genes in this leukemia group, including TCF7 which maps within the 5q31 common deleted region. Mutations of genes known to be associated with immature/early T-cell precursor acute lymphoblastic leukemia, i.e. WT1, ETV6, JAK1, JAK3, and RUNX1, were present, while CDKN2A/B deletions/mutations were never detected. All patients had relapsed/resistant disease and blasts showed an early differentiation arrest with expression of myeloid markers. Terminal 5q deletions, found in 18 of patients, were more prevalent in adults (chi-square, P=0.010) and defined a subgroup of HOXA-positive T-cell acute lymphoblastic leukemia characterized by 130 up- and 197 down-regulated genes. Down-regulated genes included TRIM41, ZFP62, MAPK9, MGAT1, and CNOT6, all mapping within the 1.4 Mb common deleted region at 5q35.3. Of interest, besides CNOT6 down-regulation, these cases also showed low BTG1 expression and a high incidence of CNOT3 mutations, suggesting that the CCR4-NOT complex plays a crucial role in the pathogenesis of HOXA-positive T-cell acute lymphoblastic leukemia with terminal 5q deletions. In conclusion, interstitial and terminal 5q deletions are recurrent genomic losses identifying distinct subtypes of T-cell acute lymphoblastic leukemia.

Background A deregulated CRLF2 (d- CRLF2 ) expression was described in B-cell acute lymphoblastic leukemia without recurrent fusion genes (B-NEG ALL). While the role of d- CRLF2 in children has been extensively described, little is known about its role and impact in adult ALL. Methods Expression levels of CRLF2 were evaluated by quantitative real-time PCR in 102 newly-diagnosed adult B-NEG ALL and correlated with the clinico-biological characteristics and outcome. Incidence and clinical impact of the P2RY8/CRLF2 transcript was also assessed. Results High CRLF2 levels, as continuous variable, were significantly associated with hyperleucocytosis ( p = 0.0002) and thrombocytopenia ( p = 0.005); when a cut-point at ΔCt ≤ 8 was applied, 35 cases (34.3%), mostly males (80%), proved positive for CRLF2 expression. High CRLF2 levels, as continuous or categorical variable, were associated with a worse disease-free ( p = 0.003 and p = 0.015) and overall survival ( p = 0.017 and 0.0038). Furthermore, when CRLF2 was analyzed as a categorical variable, a high statistical association was found with IKZF1 deletion and mutations in the JAK/STAT pathway ( p = 0.001 and p P2RY8/CRLF2 transcript, identified in 8/102 patients (7.8%), was associated with a poor outcome. Conclusions In adult B-NEG ALL, high CRLF2 expression is associated with distinct clinico-biological features and an unfavourable prognosis in both univariate and multivariate analysis; similarly, P2RY8/CRLF2 positivity correlates with a poor outcome. The quantification of CRLF2 is an important prognostic marker in adult B-lineage ALL without known genetic lesions.

Aberrant signal transduction contributes substantially to leukemogenesis. The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor proliferation, survival, and differentiation. We report that somatic mutations in JAK1 occur in individuals with acute lymphoblastic leukemia (ALL). JAK1 mutations were more prevalent among adult subjects with the T cell precursor ALL, where they accounted for 18% of cases, and were associated with advanced age at diagnosis, poor response to therapy, and overall prognosis. All mutations were missense, and some were predicted to destabilize interdomain interactions controlling the activity of the kinase. Three mutations that were studied promoted JAK1 gain of function and conferred interleukin (IL)-3–independent growth in Ba/F3 cells and/or IL-9–independent resistance to dexamethasone-induced apoptosis in T cell lymphoma BW5147 cells. Such effects were associated with variably enhanced activation of multiple downstream signaling pathways. Leukemic cells with mutated JAK1 alleles shared a gene expression signature characterized by transcriptional up-regulation of genes positively controlled by JAK signaling. Our findings implicate dysregulated JAK1 function in ALL, particularly of T cell origin, and point to this kinase as a target for the development of novel antileukemic drugs.

Fludarabine refractoriness (FR) represents an unsolved clinical problem of chronic lymphocytic leukemia (CLL) management. Although next-generation sequencing studies have led to the identification of a number of genes frequently mutated in FR-CLL, a comprehensive evaluation of the FR-CLL genome has not been reported. Toward this end, we studied 10 FR-CLLs by combining whole-exome sequencing and copy number aberration (CNA) analysis, which showed an average of 16.3 somatic mutations and 4 CNAs per sample. Screening of recurrently mutated genes in 48 additional FR-CLLs revealed that ~70% of FR-CLLs carry ≥1 mutation in genes previously associated with CLL clinical course, including TP53 (27.5%), NOTCH1 (24.1%), SF3B1 (18.9%), and BIRC3 (15.5%). In addition, this analysis showed that 10.3% of FR-CLL cases display mutations of the FAT1 gene, which encodes for a cadherin-like protein that negatively regulates Wnt signaling, consistent with a tumor suppressor role. The frequency of FAT1-mutated cases was significantly higher in FR-CLL than in unselected CLLs at diagnosis (10.3% vs 1.1%, P = .004), suggesting a role in the development of a high-risk phenotype. These findings have general implications for the mechanisms leading to FR and point to Wnt signaling as a potential therapeutic target in FR-CLL.

Notch2 mutations represent the most frequent lesion in splenic marginal zone lymphoma., Splenic marginal zone lymphoma (SMZL) is a B cell malignancy of unknown pathogenesis, and thus an orphan of targeted therapies. By integrating whole-exome sequencing and copy-number analysis, we show that the SMZL exome carries at least 30 nonsilent gene alterations. Mutations in NOTCH2, a gene required for marginal-zone (MZ) B cell development, represent the most frequent lesion in SMZL, accounting for ∼20% of cases. All NOTCH2 mutations are predicted to cause impaired degradation of the NOTCH2 protein by eliminating the C-terminal PEST domain, which is required for proteasomal recruitment. Among indolent B cell lymphoproliferative disorders, NOTCH2 mutations are restricted to SMZL, thus representing a potential diagnostic marker for this lymphoma type. In addition to NOTCH2, other modulators or members of the NOTCH pathway are recurrently targeted by genetic lesions in SMZL; these include NOTCH1, SPEN, and DTX1. We also noted mutations in other signaling pathways normally involved in MZ B cell development, suggesting that deregulation of MZ B cell development pathways plays a role in the pathogenesis of ∼60% SMZL. These findings have direct implications for the treatment of SMZL patients, given the availability of drugs that can target NOTCH, NF-κB, and other pathways deregulated in this disease.

The genetic lesions identified in chronic lymphocytic leukemia (CLL) do not entirely recapitulate the disease pathogenesis and the development of serious complications, such as chemorefractoriness. While investigating the coding genome of fludarabine-refractory CLL, we observed that mutations of SF3B1, encoding a splicing factor and representing a critical component of the cell spliceosome, were recurrent in 10 of 59 (17%) fludarabine-refractory cases, with a frequency significantly greater than that observed in a consecutive CLL cohort sampled at diagnosis (17/301, 5%; P = .002). Mutations were somatically acquired, were generally represented by missense nucleotide changes, clustered in selected HEAT repeats of the SF3B1 protein, recurrently targeted 3 hotspots (codons 662, 666, and 700), and were predictive of a poor prognosis. In fludarabine-refractory CLL, SF3B1 mutations and TP53 disruption distributed in a mutually exclusive fashion (P = .046). The identification of SF3B1 mutations points to splicing regulation as a novel pathogenetic mechanism of potential clinical relevance in CLL.

BACKGROUND: Despite recent improvements in the treatment of acute lymphoblastic leukemia (ALL), adult patients still have an overall poor outcome. The future of ALL management relies on the introduction of novel targeted therapies. The authors sought to assess if protein kinases (PKs), frequently deregulated in cancer, show an altered expression pattern and can be considered as suitable therapeutic targets in adult ALL. METHODS: The authors studied the PK gene expression profile by oligonucleotide arrays in 133 adult ALL samples at the onset of the disease and subsequently performed in vitro experiments to evaluate the sensitivity to first- and second-generation PK inhibitors of a set of ALL cell lines, as well as of primary ALL cells. RESULTS: The study documents a distinctive PK signature for different adult ALL subgroups; the PKs identified include several tyrosine kinase (TK) genes, especially in E2A/PBX+ B-lineage ALL (B-ALL), B-ALL without known molecular abnormalities, and T-lineage ALL. Consistently, cell lines and primary samples belonging to these groups proved susceptible to TK inhibitors. CONCLUSIONS: These results indicate that second-generation TK inhibitors may be effective in ALL subsets other than BCR/ABL+ B-ALL and provide the rationale for testing the impact of the newly developed TK inhibitors in the management of adult ALL patients. Cancer 2010. © 2010 American Cancer Society.

Background Until recently, few molecular aberrations were recognized in acute lymphoblastic leukemia of T-cell origin; novel lesions have recently been identified and a certain degree of overlap between acute myeloid leukemia and T-cell acute lymphoblastic leukemia has been suggested. To identify novel T-cell acute lymphoblastic leukemia entities, gene expression profiling was performed and clinico-biological features were studied.Design and Methods Sixty-nine untreated adults with T-cell acute lymphoblastic leukemia were evaluated by oligonucleotide arrays: unsupervised and supervised analyses were performed. The up-regulation of myeloid genes and miR-223 expression were validated by quantitative polymerase chain reaction analysis.Results Using unsupervised clustering, we identified five subgroups. Of these, one branch included seven patients whose gene expression profile resembled that of acute myeloid leukemia. These cases were characterized by over-expression of a large set of myeloid-related genes for surface antigens, transcription factors and granule proteins. Real-time quantitative polymerase chain reaction analysis confirmed over-expression of MPO, CEBPA, CEBPB, GRN and IL8. We, therefore, evaluated the expression levels of miR-223, involved in myeloid differentiation: these cases had significantly higher levels of miR-223 than had the other cases of T-cell acute lymphoblastic leukemia, with values comparable to those observed in acute myeloid leukemia. Finally, these patients appear to have an unfavorable clinical course.Conclusions Using gene profiling we identified a subset of adult T-cell acute lymphoblastic leukemia, accounting for 10% of the cases analyzed, which displays myeloid features. These cases were not recognized by standard approaches, underlining the importance of gene profiling in identifying novel acute leukemia subsets. The recognition of this subgroup may have clinical, prognostic and therapeutic implications.

In patients with T-cell lymphoblastic leukemia (T-ALL) chromosome rearrangements and gene mutations are used as diagnostic markers for genetic classification and prognostic stratification.[1][1] Gene deregulation in T-ALL is determined by ectopic or over-expressed oncogenes, gain or loss of function

B-cell prolymphocytic leukemia and chronic lymphocytic leukemia have distinctive gene expression signatures

Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course. To investigate the association between clinicobiologic features and responsiveness of CLL cells to anti-IgM stimulation, we evaluated gene expression changes and modifications in cell-cycle distribution, proliferation, and apoptosis of IgVH mutated (M) and unmutated (UM) samples upon BCR cross-linking. Unsupervised analysis highlighted a different response profile to BCR stimulation between UM and M samples. Supervised analysis identified several genes modulated exclusively in the UM cases upon BCR cross-linking. Functional gene groups, including signal transduction, transcription, cell-cycle regulation, and cytoskeleton organization, were up-regulated upon stimulation in UM cases. Cell-cycle and proliferation analyses confirmed that IgM cross-linking induced a significant progression into the G1 phase and a moderate increase of proliferative activity exclusively in UM patients. Moreover, we observed only a small reduction in the percentage of subG0/1 cells, without changes in apoptosis, in UM cases; contrariwise, a significant increase of apoptotic levels was observed in stimulated cells from M cases. These results document that a differential genotypic and functional response to BCR ligation between IgVH M and UM cases is operational in CLL, indicating that response to antigenic stimulation plays a pivotal role in disease progression.

Among all cytogenetic subtypes of ALL, the BCR-ABL1 fusion gene, which is causative of chronic myeloid leukemia (CML), defines the subgroup of ALL with the worst prognosis. The reasons of the aggressive nature of BCR-ABL1-positive ALL have not yet been completely understood. To identify, at the submicroscopic level, oncogenic lesions that cooperate with BCR-ABL1 to induce ALL and blastic transformation of CML, we used an integrated molecular approach including high resolution genomic analysis of copy number alterations by single nucleotide polymorphism (SNP) arrays (500K, Affymetrix Inc., Santa Clara, CA, USA), genomic amplification, cloning, deep sequencing, gene expression profiling (GEP) and analysis of epigenetic changes to study 97 de novo BCR-ABL1- positive ALL adult patients (pts). High level amplification and homozygous deletions were identified in all pts, with deletions outnumbering amplification almost 3:1. The most frequent somatic copy number alteration was deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros identified in 59/97 pts (61%). Ikaros is required for the earliest stages of lymphoid lineage commitment and acts as tumor suppressor in mice. The IKZF1 deletions were predominantly mono-allelic (64% vs 36%) and were limited to the gene in all cases, identifying IKZF1 as the genetic target. Real-time quantitative PCR and FISH analysis confirmed SNP results. We characterized and mapped all breakpoints to recognize that two major deletions occur in BCR-ABL1-positive ALL: type A characterized by loss of exons 4–7 (37/97, 38%) and due to breakpoints occurring in the region spanning from 50380371 to 50380388 and from 50431123 to 50431167 in introns 3 and 7, respectively; type B characterized by removal of exons 2–7 (18/97, 19%) and due to a variable pattern of breakpoints in introns 1 (from 50317112 to 50317936) and 7. A variable number of nucleotides (patient-specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of both deletions correlated with the expression of a dominant-negative isoform Ik6 with cytoplasmic localization and oncogenic activity, and of an aberrant transcript containing only exons 1 and 8, respectively. Interestingly, 2 pts with a homozygosis deletion showed simultaneously both type A and B deletions. In other 2 pts, the deletion involved the whole IKZF1 gene. The IKZF1 alteration was not a frequent event across different leukemias, since it was identified also at the progression of CML to lymphoid blast crisis (66%), but never in myeloid blast crisis CML (n=10), chronic phase CML (n=30) and acute myeloid leukemia (n=28). Known DNA sequences and structural features were mapped along the breakpoint cluster regions including heptamer recombination signal sequences recognized by the RAG enzymes during V(D)J recombination and activation-induced cytidine deaminase (AID) consensus motifs (DGYW/WRCH), suggesting that IKZF1 deletions could arise from aberrant RAG and/or AID-mediated recombination. GEP analysis of pts with IKZF1 deletion vs wild-type pts identified a unique signature characterized by a down-regulation of genes involved in pre-B-cell differentiation (e.g. VPREB1, VPREB3, IGLL3), demonstrating that genomic IKZF1 alterations have a strong impact on trascriptoma and contribute to a block of B-cell differentiation. For 83 out of 97 BCR-ABL1 ALL pts correlation between molecular data and clinical outcome was available. Univariate analysis showed that the IKZF1 deletion is a negative prognostic marker influencing the cumulative incidence of relapse (10.1 months for pts with deletion vs 56.1 months for wild-type pts, p=0.0103) and disease-free survival (DFS) (10.1 months vs 32.1 months, respectively, p=0.0229). The negative prognostic impact of the IKZF1 deletion on DFS was also confirmed by multivariate analysis (p=0.0445). In conclusion, deletion of IKZF1 resulting in either haploinsuffciency and in the expression of the dominant negative isoforms is an important event in the development of BCR-ABL1 B-progenitor ALL which significantly influences clinical outcome.

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 IgVH 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.

Background and Objectives Recent data have highlighted an involvement of ABL1 in T-cell acute lymphoblastic leukemia (T-ALL). Specifically, the presence of a fusion gene involving ABL1 and NUP214 , both located at 9q34, has been reported. We sought to evaluate whether TALL patients with overexpression of ABL showed a peculiar gene expression pattern and were characterized by having specific rearrangements. Design and Methods We previously assessed the expression profile of 128 adults with ALL by oligonucleotide arrays: 33 had T-ALL. In the current study, we evaluated the expression levels of ABL1 in T-ALL cases and found three patients who had ABL1 levels comparable to those detected in BCR/ABL+ cases and one who had a significantly higher level of ABL1 expression. In order to establish the incidence of ABL1 overexpression in TALL, we evaluated 17 additional patients by quantitative (Q)-polymerase chain reaction (PCR) and reverse transcription (RT)-PCR. Results The three cases with ABL1 expression levels comparable to those found in BCR/ABL+ cases had a specific signature characterized by a high expression of genes involved in regulation of transcription. The fourth case, with the highest levels of ABL1 , harbored the NUP214-ABL1 rearrangement, which was confirmed by fluorescence in situ hybridization (FISH). Three of the four patients were refractory to induction chemotherapy. Of the 17 additional patients evaluated by Q-PCR and RT-PCR, none showed ABL1 overexpression. Interpretation and Conclusions Overall, overexpression of ABL1 was found in 8% of T-ALL cases. These results underline the value of microarray analyses for the identification of specific signatures associated with ABL1 overexpression, as well as rearrangements, e.g. NUP214-ABL1 , in adult T-ALL.

Background. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of relatively mature B cells and by a very variable clinical course. This clinical heterogeneity is sustained by different biologic parameters, such as the mutational status of the immunoglobulin variable genes (IgVH), CD38 and ZAP-70 expression. In order to investigate the potential role of protein kinase (PK) inhibitors in CLL, we evaluated the gene expression profile of 1324 probesets annotated as PK using the HGU133 Plus2.0 Affymetrix arrays. Methods. We evaluated 44 CLL and 137 acute lymphocytic leukemia (ALL) patients. Two additional sets of CLL (49 cases) were utilized to validate the results obtained. Probesets identified as PK genes were used for all the analyses, namely unsupervised clustering, Analysis of Variance (ANOVA) and t-test analysis. ANOVA was performed using a p-value of ≤0.001: further selection was performed retaining only those probesets whose mean expression level was ≥300 in at least one group and showed a fold change difference of ≥1.5 across all groups. Finally, to specifically identify genes differentially expressed between different subclasses of CLL, a t-test was applied: probesets were required to have a p-value ≤0.05 and a fold change>1.5. Results. Unsupervised analysis, performed on CLL samples and different ALL subgroups, highlighted in CLL a unique and very homogeneous pattern characterized by the overexpression of a large set of PK; these results were further confirmed by ANOVA. Moreover, we identified 16 PK genes that were highly expressed in all 3 CLL sets analyzed. These genes codify for proteins with tyrosine kinase activity (SYK, LYN, BLK, LCK, JAK1, CSK and FGR), serin-threonin kinase activity (PIM2, PFTK1, TLK1, MAP4K1, PDPK1, PRKCB1 and STK10) or both (GRK6 and WEE1). Some of the selected genes are members of important protein kinase families, involved in cellular signaling, such as Src kinases (SFK), MAPK and JAK kinase family. PK expression was also analyzed in different CLL subclasses, subdivided according to different prognostic factors; in particular, we compared IgVH mutated vs unmutated patients, CD38+ vs CD38- cases and, finally, ZAP-70+ vs ZAP-70- patients in the 3 experimental CLL sets. Comparison between IgVH mutated vs unmutated cases highlighted a differential expression of ZAP-70 in all the 3 sets analyzed. Contrariwise, no PK was associated with the other prognostic parameters. Thus, these analyses did not show a specific signature associated with the abovementioned biologic features, suggesting that PK overexpression is specific of the disease itself rather than of CLL subclasses. Conclusions. Our results show that CLL is characterized by a very peculiar PK signature and identify some potential molecular targets. Moreover, our findings indicate that a common mechanism of PK-mediated deregulation is operational in CLL cells, independently of other prognostic factors. Based on these pre-clinical data, we propose that second generation PK inhibitors may have a role in the management of all CLL patients.

Abstract 1784 Introduction: The introduction of whole exome sequencing has allowed to unravel novel molecular lesions in CLL. NOTCH1, SF3B1 and BIRC3 mutations are detected, according to the phases of disease, in 4–12%, 5–17% and 4–24% of patients, respectively. In retrospective studies, their presence has been shown to correlate with overall survival (OS) and treatment-free interval shortening. Aims: To define the incidence, correlation with known prognostic factors and clinical impact of NOTCH1, SF3B1 and BIRC3 mutations in CLL patients undergoing first-line treatment. Methods: We evaluated 162 CLL patients enrolled in the GIMEMA LLC0405 protocol (n=80) for patients aged 65 yrs or 60–65 if not eligible for fludarabine-based programs). In the GIMEMA LLC0405 protocol, patients were stratified into low and high-risk: patients with del17p or with del11q plus an unmutated IGHV status and/or CD38 positivity and/or ZAP70 positivity were considered as high-risk (HR) and underwent Fludarabine plus Campath, followed by stem cell transplantation procedures, whereas low-risk patients received Fludarabine and Cyclophosphamide. The MLL21445 protocol consisted of 8 cycles of Chlorambucil and 6 of Rituximab induction treatment. NOTCH1 (exon 34), SF3B1 (exons 14 and 15) and BIRC3 (exons 2–9, including splicing sites) were screened by Sanger sequencing on either genomic DNA (gDNA) or whole genome amplified DNA (WGA) collected at the time of treatment. These studies were not part of the clinical protocols. Results: NOTCH1 mutations were detected at the time of treatment in 18 cases (22%) enrolled in the LLC0405 study. There was a significant association with high-risk stratification (p=0.036), namely with an IGHV unmutated status (p=0.0035), CD38 (p=0.03), +12 (p=0.034) and, partly, ZAP-70 expression (p=0.059). While the overall response rate (ORR) did not differ between NOTCH1 mutated vs wild-type (WT) cases (82% vs 77%, respectively), the complete response (CR) rate was significantly lower in NOTCH1 mutated patients (43% for WT vs 17% for NOTCH1 mutated cases; p=0.05). So far, no significant difference between mutated and WT patients has emerged in terms of OS and progression-free survival (PFS); this may be contributed by the fact that most NOTCH1 mutated cases were HR and were therefore treated more aggressively. SF3B1 mutations were recorded in 9 cases (11%); no significant associations were found with known biological parameters and, so far, with the ORR and CR rate. A single case harbored a BIRC3 mutation; this patient had an IGHV unmutated status, no FISH abnormalities and a concomitant SF3B1 mutation. In the ML21445 cohort, NOTCH1 mutations were found in 12 cases (15%), were associated with an unmutated IGHV status (p=0.047) and ZAP-70 expression (p=0.007), and did not impact on the ORR and CR rate. SF3B1 mutations were found in 11 cases (13%); no significant associations were found with known biological parameters and the ORR rate. Of interest, only 1/11 SF3B1 mutated patients achieved a CR. BIRC3 mutations were recorded in 3 patients (3.6%); of these, 2 were IGHV mutated, 1 had no cytogenetic abnormalities and 1 carried a del11q, while the third patient was IGHV unmutated status and had no cytogenetic abnormalities. No NOTCH1 and/or SF3B1 mutations were detected. Overall, NOTCH1, SF3B1 and BIRC3 mutations were largely mutually exclusive among each other and with TP53 lesions in the whole cohort. Conclusions: This study confirms the association of NOTCH1 mutations with unfavorable biologic markers and +12, while the presence of SF3B1 mutations was not coupled to poor prognostic markers in CLL patients requiring first-line treatment. Furthermore, it suggests that NOTCH1 mutations impact on the CR rate of young patients receiving Fluda-based regimens, while SF3B1 appears to impact on the CR rate of elderly patients treated with Chlorambucil and Rituximab. Given the small numbers of patients harboring BIRC3, it is at present difficult to draw any conclusion on the clinical impact of this mutation in the cohort of patients hereby analyzed. Disclosures: No relevant conflicts of interest to declare.

Abstract 464 Fludarabine-refractoriness of chronic lymphocytic leukemia (CLL) is due to TP53 disruption in ∼40% of refractory cases, but in a sizeable fraction of patients the molecular basis of this aggressive clinical phenotype remains unclear. Our initial findings from whole exome sequencing of fludarabine-refractory CLL led to the identification of recurrent mutations of SF3B1, a critical component of the cell spliceosome, prompting further investigations of these alterations in a large CLL panel. The study population comprised 3 clinical cohorts representative of: i) fludarabine-refractory CLL (n=59), including cases (n=11) subjected to whole exome sequencing; ii) newly diagnosed and previously untreated CLL (n=301); and iii) clonally related RS (n=33). Tumor samples were obtained: i) for fludarabine-refractory CLL, immediately before starting the treatment to which the patient eventually failed to respond; ii) for newly diagnosed and previously untreated CLL, at disease presentation. All RS studies were performed on RS diagnostic biopsies. Mutation analysis of SF3B1 was performed on genomic DNA by a combination of Sanger sequencing and targeted next generation sequencing. SF3B1 was altered in 10/59 (17%) fludarabine-refractory CLL by missense mutations (n=9) or in-frame deletions (n=1) clustering in the HEAT3, HEAT4 and HEAT5 repeats of the SF3B1 protein. Two sites that are highly conserved inter-species (codon 662 and codon 700) were recurrently mutated in 3 and 5 cases, respectively. SF3B1 mutations were monoallelic, and were predicted to be functionally significant according to the PolyPhen-2 algorithm. Mutations occurred irrespective of IGHV mutation status, CD38 expression and ZAP70 expression. At the time of fludarabine-refractoriness, SF3B1 mutations were enriched in cases harboring a normal FISH karyotype (p=.008) and distributed in a mutually exclusive fashion with TP53 disruption (mutual information I =0.0609; p=.046). By combining SF3B1 mutations with other genetic lesions enriched in chemorefractory cases (TP53 disruption, NOTCH1 mutations, ATM deletion), fludarabine-refractory CLL appeared to be characterized by multiple molecular alterations that, to some extent, are mutually exclusive. We then compared the prevalence of mutations observed at the time of fludarabine-refractoriness to that observed in other disease phases. At diagnosis, SF3B1 mutations were rare (17/301; 5%), and showed a crude association with short treatment free survival (p Disclosures: No relevant conflicts of interest to declare.

Purpose. CLL is a malignancy of mature B cells with an heterogeneous clinical course. In order to identify novel therapeutic targets and given the role of protein kinase (PK) deregulation in cancer development and the availability of specific PK inhibitors, oligonucleotide arrays were used to determine if CLL patients exhibit a specific PK pattern. The results were validated by Q-PCR, and the in vitro efficacy of a dual specific inhibitor, dasatinib (Bristol-Myers Squibb, Princeton, NJ), was tested. Methods. The gene expression profile of 44 CLL and 137 acute lymphocytic leukemia (ALL) patients was evaluated using the HG U133 Plus 2.0 Affymetrix arrays. Two additional sets of CLL (49 cases) were utilized as test sets: 505 PK genes were used for all analyses, which included unsupervised clustering, Analysis of Variance (ANOVA) and t-test analysis. To validate the gene expression data, a Q-PCR approach was used on 14 CLL, 6 B-lineage ALL, 3 T-ALL and CD19+ enriched normal B cells, isolated from peripheral blood of 3 healthy donors. Finally, to evaluate the in vitro effects of dasatinib on primary CLL cells, the MTT assay was performed on 21 untreated CLL samples following 72 and 96 hours of drug exposure. Results. Unsupervised analysis on CLL samples and different ALL subgroups showed a very homogeneous PK gene profile in CLL. ANOVA corroborated these results. Among the PK that proved overexpressed in CLL, we identified 16 PK genes highly expressed in all 3 CLL sets analyzed. For these genes, Q-PCR analysis showed that CLL cases display a significantly higher expression level, when compared to B-lineage ALL, T-ALL and, more importantly, to CD19+ cells from healthy controls. PK expression was also analyzed within different CLL subclasses, subdivided on the basis of the IgVH mutational status, as well as CD38 and ZAP-70 expression. The comparison among these groups did not show a specific PK signature associated with biologic features; similar results were obtained by Q-PCR analysis. To validate the gene expression profiling and Q-PCR findings, and to test the efficacy of dasatinib, functional in vitro experiments were performed on primary CLL cells. Treatment with 1μM dasatinib induced an overall viability reduction of 40% after 96 hours in the CLL samples analyzed; no significant differences were associated with the IgVH mutational status. These findings indicate that this inhibitor is functionally active on CLL cells, without however reaching marked levels of cytotoxicity when used as a single agent. Conclusions. Our results highlight a very homogeneous PK signature in CLL, independently of specific biologically-defined prognostic subgroups. A set of these PKs is more highly expressed in CLL cells than in CD19+ cells from healthy donors. Dasatinib treatment induces a ∼40% viability reduction after 96 hours exposure. Overall, these results suggest that PK inhibitors, namely dual kinase inhibitors, may have a role in the management of CLL patients particularly in combination with other therapeutic agents. * ST and SC equally contributed to the study

Background. Despite the recent improvements in the treatment of acute lymphocytic leukemia (ALL), adults still have a poor outcome. The future of ALL management is likely to rely in the progressive introduction of novel targeted therapies. Protein kinase (PK) deregulation has been frequently observed in cancer; therefore, PK are regarded as attractive targets for anti-tumor therapies. The majority of PK inhibitors currently used in clinical trials in onco-hematology targets tyrosine kinases (TK) and receptor tyrosine kinases (RTK). Methods. We evaluated the expression levels of PK genes in 133 adult ALL samples at the onset of the disease by oligonucleotide arrays (HGU133 Plus 2.0, Affymetrix). Leukemic cells from 91 samples were of B-cell origin and belonged to 4 molecular subgroups: BCR/ABL+ (40 pts), ALL1/AF4+ (5 pts), E2A/PBX+ (3 pts) and B-lineage ALL without known molecular abnormalities (43 pts), defined as B-NEG. The remaining 42 samples were of T-cell derivation. To compare the 5 ALL subgroups we performed an ANOVA analysis using a list of 1324 probesets coding for PK. The genes identified by ANOVA were further selected (mean expression values ≥300, fold change difference ≥1.5 in each group compared to every other group) to concentrate on distinguishing PK genes. This approach identified a set of distinctive PK for each ALL subgroup except for the B-NEG samples. To further investigate the B-NEG group we compared it with every other B-lineage ALL subset by t test using the aforementioned gene list and highlighted the genes that were overexpressed in this group when compared to at least two other subgroups. Results. ANOVA identified 290 probesets differentially expressed among the 5 ALL subgroups. The selection criteria applied allowed to identify the PK genes distinctive of each group and to highlight those of particular interest, as TK and RTK. T-ALL and E2A/PBX+ ALL were characterized by the specific expression of 18 PK genes. T-ALL showed a high expression of ZAP-70, LCK, ITK, EPHB6, FGFR1 and RYK. MERTK, BLK, TNK2 were distinctive of the E2A/PBX+ group. Fifteen genes were specific of the ALL1/AF4+ samples: among these, FLT3, ILK and LTK were selected. Only 8 genes were specifically overexpressed in the BCR/ABL+ samples; apart from ABL, this group showed high expression levels of FYN. Although no PK was exclusively upregulated in the B-NEG cases, a consistent number of PK genes was overexpressed in these samples. Conclusions. Our study indicates that each ALL subgroup shows a distinctive PK signature. The specific PK subsets identified include several TK and RTK genes - especially in T-ALL, and in the E2A/PBX+ and B-NEG groups - whose products may be suitable for targeted therapies. These results suggest that second generation TK inhibitors may be effective not only in BCR/ABL+ patients, but also in other ALL subsets, such as T-ALL, e2a/pbx+ and B-NEG ALL, and provide the rationale for testing the impact of currently available TK inhibitors in the management of adult ALL.

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.

Background. CLL is the most common leukemia in the western world and is characterized by the accumulation of relatively mature B cells. From a clinical point of view, CLL patients display a variable course. This clinical heterogeneity is sustained by different biologic parameters, such as the immunoglobulin variable gene (IgVH) mutational status, ZAP-70 expression and specific cytogenetic alterations. Even though extensive molecular characterizations of CLL cells have been performed, a candidate gene responsible for the disease is still lacking. Thus, there is a compelling need to identify other mechanisms that may play a role in CLL. miRs are an abundant class of small non-coding RNAs which modulate the expression of their target mRNAs at the post-transcriptional and/or translational level. So far, 462 miRs have been identified in the human genome: many are involved in cancer, acting either as oncogenes or tumor suppressors. Methods. In order to define the potential role of miRs in CLL, the expression of these small RNAs was evaluated by cloning, as well as by quantitative Real-Time PCR (qRT-PCR) in a total of 56 CLL patients: 9 cases were evaluated by cloning, 46 by qRT-PCR and 4 by both methods; peripheral blood B lymphocytes and cord blood cells enriched for CD19+ were used as controls. Results. By cloning, it was possible to identify roughly 20 miRs that contribute to more than 90% of the expressed miRs in CLL and healthy donors. Furthermore, we observed a significant upregulation (at least 3 fold) of miR-21, miR-155 and miR-150 in CLL patients compared to healthy donors. To validate these findings, we next measured the expression of 22 of the most expressed miRNAs in 51 CLL patients and in 7 healthy donors (5 CD19+ peripheral blood lymphocytes and 2 CD5+ cord blood cells) by qRT-PCR: this approach confirmed the differential expression of miR-21, miR-155 and miR-150 expression between leukemic and normal cells, and identified 2 additional miRs, namely miR-92 and miR-222, that are downregulated in the controls. Unsupervised clustering based on qRT-PCR results allowed to subdivide the cases analyzed into 3 major subgroups: the first comprised all the 5 controls, the second a set of patients that had a strong downregulation of miR-15 and miR-16, and the third was inclusive of all the remaining cases. It is of worth noting that the patients who showed a downregulation of miR-15 and miR-16 had a del13q14 in homozygosis, in line with previous reports. Finally, a supervised approach highlighted a differential expression of miR-150, miR-29bc and miR-223 between IgVH mutated and unmutated; consistently with this finding, miR-150 was also differentially expressed between ZAP-70+ and ZAP-70− cases. Conclusions. Our study indicates that 3 miRs are strongly and homogeneously upregulated in CLL patients, suggesting that they may play an important role in disease initiation and/or progression. Finally, a small set of miRs is distinctive of prognostic subgroups. Further investigations on these small RNAs is ongoing, to specifically define their role in CLL.

In adult patients with acute lymphoid leukemia (ALL) conventional therapies can cure only about 20% of patients. Despite the high proportion of complete remissions, the prognosis remains poor because the disease is not eradicate and patients eventually relapse. It is thus important to develop new therapeutic strategies. Dendritic cell (DC)-based vaccines have been tested in different neoplastic conditions, including patients with hematological malignancies, and the possibility of eliciting specific responses has been documented. In this study, we evaluated in adult ALL patients in complete remission and off-therapy the possibility of generating DC (ALL-DC) and the ability of ALL-DC to stimulate allogeneic and autologous anti-leukemic T lymphocytes. In all 15 B-lineage and 2 T-lineage ALL cases studied it has been possible to generate adequate numbers of DC, i.e. 1–1.5 x 106 from 20 ml of peripheral blood. After gradient separation and adhesion, monocytes were incubated with granulocyte/macrophage colony-stimulating-factor (GM-CSF) and interleukin-4 (IL-4) for 5 days, and tumor necrosis factor (TNF)-α for 48 hours. The expression of the DC markers CD1a, CD80, CD86, CD83 and CD40, evaluated by flow cytometry on ALL-DC, was comparable with that observed on DC derived from 10 normal donors. ALL-DC were capable of loading apoptotic autologous and allogenic leukemic bodies generated in vitro after incubation with Imatinib (for Bcr/Abl+ ALL) or Fludarabine. Successful loading has been demonstrated by flow cytometry utilizing the PKH67 fluorescent dye incubated with the leukemic cells before drug treatment. In all patients, the T-lymphoid population showed a proliferative capacity and an IFNγ and TNFα intracytoplasmic production, after PHA and PMA stimulation, comparable to that generated in lymphocytes from normal donors. Furthermore, in 6/7 B-lineage ALL patients tested DC-pulsed with apoptotic leukemic cells were capable of stimulating the proliferation of both allogenic and autologous lymphocytes. We also studied 4 further patients affected by B-lineage ALL who carried the molecular alteration Bcr/Abl. These patients were treated daily with Imatinib and at the time of the study were in complete morphologic remission. T lymphocytes, repeatedly evaluated, showed a normal proliferative response to PHA stimulation. The generation of DC has proven numerically and phenotypically comparable to that of normal donors, but in 2/3 cases analyzed DC showed a reduced ability to load apoptotic leukemic cells. In conclusion, the results of this study indicate that DC can be effectively generated in vitro from adult ALL patients at the time of complete remission. These DC pulsed with leukemic apoptotic bodies are capable of stimulating an anti-leukemic T-lymphocyte response, also under autologous conditions. Our data suggest that in ALL patients in complete remission of their disease, an immunotherapeutic strategy utilizing DC could have a role in the control of the leukemia. In Bcr/Abl+ patients treated with Imatinib, further studies are necessary to fully define the competence of the DC compartment.

Purpose. CLL is an heterogeneous disease with a variable clinical course. In order to investigate the association between specific clinico-biological features and the ability of CLL cells to respond to anti-IgM-mediated signaling, we evaluated the gene expression changes upon BCR stimulation as well as the changes in cell cycle, proliferation and apoptotic rate of IgVH mutated and unmutated samples. Methods. After 24 hours incubation with a F(ab)2 anti-m antibody (10 mg/ml), unstimulated (US) and stimulated (S) CD19+ B cells isolated from untreated CLL patients underwent microarrays analysis using the HGU133 Plus 2.0 Affymetrix arrays. Unsupervised clustering and t-test analyses were performed. In addition, Q-PCR analysis was carried out to evaluate the levels of SYK and ZAP-70 expression in CLL samples at different time points (6 and 24 hours) upon BCR ligation. At 24, 48 and 72 hours from the stimulus, cell cycle distribution changes were evaluated using the Acridine Orange (AO) technique, cell proliferation was measured by 3H-TdR uptake and apoptosis was analyzed by the Annexin-V and/or AO technique. Results. Unsupervised analysis on CLL samples showed that response to BCR stimulation is strictly associated to the IgVH mutational status and IgM expression levels. Based on these findings, to specifically identify the genes that were modulated upon BCR ligation, we performed a t-test to compare US and S samples within germline and mutated cases. In the germline cases, this analysis identified 197 genes differentially expressed; among the more represented functional groups, we found several genes involved in signal transduction, regulation of transcription, cell cycle regulation as well as cytoskeleton. Contrariwise, using the same approach for the mutated cases, no genes were selected in this analysis, suggesting that BCR stimulation induces relevant changes exclusively on IgVH germline patients. To investigate the effects of IgM cross-linking on BCR signaling, we evaluated SYK and ZAP-70 expression in US and S CLL samples by Q-PCR approach. These studies showed that SYK, but not ZAP-70, is down-modulated upon stimulus only in germline cases. Furthermore, cell cycle analysis and proliferation assay documented that IgM cross-linking at 48 hours induced a significant progression into the G1-phase (p=0.037) and a moderate increase of proliferative activity in CLL cells exclusively in germline patients. Moreover, at the same time point we observed only a partial reduction of the percentage of subG0/1 cells without changes in apoptosis in CLL germline cases; contrariwise, increased levels of apoptosis (p=0.04) were observed in S cells from CLL mutated cases. Conclusions. Gene expression profile highlights a different responsiveness to BCR stimulation between IgVH germline and mutated CLL samples. In line with these results, in vitro experiments have shown that differences in cell cycle distribution, proliferative activity and apoptosis levels upon BCR ligation correlate with the IgVH mutational status of the CLL samples, supporting the hypothesis that response to BCR-ligation may play a crucial role in disease progression in IgVH germline cases. * ST and RM equally contributed to the study

Introduction. Chronic lymphocytic leukemia (CLL) with 11q deletion has been associated to a poor prognosis, but the clinical course of patients carrying this lesion is variable. This aberration, most often monoallelic, is present in 10-17% of newly diagnosed CLL and in 20-30% of patients with progressive or chemorefractory disease. The minimal deleted region (MDR) (2-3 Mbp) is located on the 11q22.3-q23.1 region and includes ATM. Moreover, 30-40% of 11q- CLL have also an inactivating ATM mutation on the other allele. The deleted region on 11q can also include BIRC3, a gene that is often deleted or mutated in advanced/chemorefractory stages of the disease. Although BIRC3 disruption has been associated to a poor prognosis, its prognostic implications in addition to ATM deletion are not well defined. The aim of this study was to perform a copy number aberration (CNA) and gene sequencing analyses on a cohort of CLL patients with 11q- in order to identify subgroups with potential prognostic relevance based on: i) the inclusion of BIRC3 in the deleted region; ii) the presence of BIRC3 mutation; iii) the presence of other CNAs. Methods. The study has included 55 untreated CLL patients followed at our Institution or enrolled in GIMEMA clinical trials (2003-2013). Genomic DNA was extracted from peripheral blood samples. CNA analysis was performed by genomic hybridization on the CytoScan HD array (Affymetrix), which contains more than 2.6 x 106 markers for copy number analysis and 750.000 SNPs. Data were analyzed using both Partek Genomics Suite and ChAS (Chromosome Analysis Suite, Affymetrix) software. The resulting CNAs were verified by visual examination of the plotted copy number profiles. BIRC3 mutations (exons 6-9) were evaluated by Sanger sequencing. Time to first treatment (TFT) was calculated from the date of diagnosis to the date of first therapy or last follow-up; progression-free survival (PFS) from the date of first therapy to the date of progression, death or last follow-up. Results. Baseline characteristics of the 55 cases were as follows: median age at diagnosis 59 years (range 39-84), male gender in 81.8% of patients, progressive disease in 62%. All patients showed 11q- by FISH (median 80%, range 25-99% of nuclei); germline IGHV were present in 96.4% of cases; TP53 deletion in 1 case and TP53 mutation in none; NOTCH1 mutation in 4/40 cases; SF3B1 mutation in 5/40 (all mutually exclusive with only 1 case having both SF3B1 and BIRC3 mutations). By CytoScan HD array, the size of 11q- was very variable, ranging from 0.36 Mbp to 65.14 Mbp; the MDR was located on 11q22.3 region, encompassing 4 genes (ACAT1, ATM, CUL5, NPAT). BIRC3 was included in the deleted region in 45/55 cases (81.8%) and was mutated in 4/54 (7.5%), being always deleted on the other allele. Beside 11q-, 51 cases (92.7%) showed several additional CNAs (average 4.9, range 1-14 per patient), with 5 recurrent lesions: 2p gain in 11 cases, del4(p15.2) in 6, del19(p13.3) in 6, 8q gain in 5 and del4(q22.1) in 4. BIRC3 deletion was not associated to the number of additional CNAs nor to specific CNA. After a follow-up of 59.6 months (range 7.4-229.7), 40 of 47 evaluable patients have received treatment (median TFT 15.8 months, range 0-167). BIRC3 deleted cases (n=37) showed a TFT not significantly different from WT cases (n=10). Conversely, BIRC3 mutation was associated to a shorter TFT (p 3 CNAs larger than 5 Mb (n=14) or >10 CNAs (n=5), or the presence of 2p gain, del4(p15.2), del19(p13.3) or 8q gain. So far, 22 patients have been evaluated for PFS after first-line therapy (median 28.7 months). BIRC3 deleted cases (n=17) were not associated to a shorter PFS compared to WT cases (n=5), in line with the results from Rose-Zerilli et al (Haematologica 2014). Conclusions. Among CLL with 11q-: 1) BIRC3 deletion involves more than 80% of cases, whilst the mutation is rare (7.5%); 2) BIRC3 deletion is not associated to a higher genomic complexity; 3) BIRC3 deletion does not seem to influence TFT or PFS of 11q- CLL; 4) BIRC3 mutation is strongly associated to a short TFT; 5) BIRC3 biallelic lesions can be associated to a marked hyperleucocytosis at diagnosis and immediate need of treatment. Disclosures No relevant conflicts of interest to declare.

Introduction: Among B-lineage ALL, BCR/ABL1-like cases have clearly emerged as a subgroup with peculiar features. By gene expression profiling (GEP), they have a transcriptional signature similar to that of BCR/ABL1+ cases. Moreover, these cases often have IKZF1 deletions and CRLF2 deregulation, and harbor several lesions involving tyrosine kinases (TKs) that may be patient specific. The incidence varies with age (from 10% of B-lineage ALL in children, to 27% in adolescents/young adults, with a decrease in adults) and they are usually characterized by an inferior outcome, though risk-directed therapy based on minimal residual disease levels has improved prognosis in childhood. Even if GEP promptly recognizes these patients, this technique is expensive, not readily available and not applicable to all centers. Aims: To generate a simple and reliable tool, based on a Q-RT-PCR approach, aimed at the rapid identification of BCR/ABL1-like cases, and to build a statistical predictive model. Finally, to correlate the results obtained with the clinico-biologic features and outcome. Methods and patients: Through a meta-analysis of previously published GEP data focused on BCR/ABL1-like cases, and on our in house results, 9 commonly overexpressed genes were selected and evaluated by Q-RT-PCR; CRLF2 was also included, because of its association with this signature (total=10 genes). Q-RT-PCR was performed using the ABI PRISM 7300 Sequence Detection System and FastStart Universal SYBR Green Master (Rox). Overall, 149 B-lineage ALL cases, negative for known major rearrangements, i.e. BCR/ABL1, ETV6/RUNX1, E2A/PBX1 and ALL1 (B-NEG ALL), were tested. Median age was 28.8 years (range 1-78), 59 were females and 90 males. To generate a predictive model for BCR/ABL1-like identification, one round of cross-validation was performed; the full dataset was divided into two panels (discovery and screening). The discovery panel included 52 cases previously evaluated for GEP analysis and comprised 26 BCR/ABL1-like and 26 B-NEG ALL samples, while the screening panel (n=97) was based on 81 cases including 16 B-NEG ALL samples harboring JAK2, IL7R or CRLF2 mutations, tested as internal control, given their association with a BCR/ABL1-like signature. Results: The statistical model was built on the discovery panel and was validated, in terms of sensitivity and specificity, on the screening panel. Q-RT-PCR values of the 10 genes were grouped in the discovery panel according to principal component analysis to reduce multicollinearity and a logistic regression model was used to examine the association among the first three principal components (accounting for more than 80% of the total variance) and BCR/ABL1-like cases. Finally, a score was computed and validated in the screening panel. The model had a sensitivity and specificity of 93.8% and 93.8%, respectively, with a positive predictive value of 75%. Within the screening panel, 10/81 (12.3%) and 6/16 JAK2, IL7R or CRLF2 mutated cases (37.5%) were predicted as BCR/ABL1-like (total=16.5%). GEP was performed on 14/16 BCR/ABL1-like samples and confirmed the similarity with BCR/ABL1+ ALL cases by unsupervised and supervised analysis. Molecular screening, comprising the screening of JAK1/2, CRLF2, IL7R (JAK/STAT pathway) mutations and IKZF1 deletions, confirmed a significant enrichment of the JAK/STAT pathway (48% vs 15%, p=0.001) and of IKZF1 deletions (82 vs 46%, p=0.006) in BCR-ABL1-like cases as opposed to the other B-NEG ALL cases. Finally, BCR-ABL1 -like patients showed a significantly higher white blood cells (WBC) count (46x109/l vs 12.8x109/l, p=0.003) and poorer prognosis compared to the other B-NEG ALL patients, in terms of complete remission achievement (70.3 vs 87%, p=0.07), overall survival (41.1% vs 59.1%, p=0.06) and disease-free survival (DFS) (31.1 % vs 46%, p=0.008). Conclusions: We describe a Q-RT-PCR approach and a statistical model capable of rapidly identifying BCR-ABL1-like cases. We confirm a significant association with JAK/STAT mutations, IKZF1 deletions and poorer outcome. The prompt recognition of BCR-ABL1-like ALL at presentation can: i) identify cases in which additional lesion/s involving a TK - that might sustain the BCR/ABL1-like signature - should be further investigated; ii) drive therapeutic decisions, since these patients might benefit by the addition of targeted therapies, particularly TK inhibitors. Disclosures Foà: Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.