ALBERT

Beschreibung: Key Points SOX11 silencing promotes the shift from a mature B cell into the initial plasmacytic differentiation phenotype in MCL. SOX11 promotes tumor growth of MCL cells in vivo, highlighting its implication in the aggressive behavior of conventional MCL.

Beschreibung: Diffuse large B-cell lymphomas (DLBCLs) can be divided into germinal-center B cell–like (GCB) and activated-B cell–like (ABC) subtypes by gene-expression profiling (GEP), with the latter showing a poorer outcome. Although this classification can be mimicked by different immunostaining algorithms, their reliability is the object of controversy. We constructed tissue microarrays with samples of 157 DLBCL patients homogeneously treated with immunochemotherapy to apply the following algorithms: Colomo (MUM1/IRF4, CD10, and BCL6 antigens), Hans (CD10, BCL6, and MUM1/IRF4), Muris (CD10 and MUM1/IRF4 plus BCL2), Choi (GCET1, MUM1/IRF4, CD10, FOXP1, and BCL6), and Tally (CD10, GCET1, MUM1/IRF4, FOXP1, and LMO2). GEP information was available in 62 cases. The proportion of misclassified cases by immunohistochemistry compared with GEP was higher when defining the GCB subset: 41%, 48%, 30%, 60%, and 40% for Colomo, Hans, Muris, Choi, and Tally, respectively. Whereas the GEP groups showed significantly different 5-year progression-free survival (76% vs 31% for GCB and activated DLBCL) and overall survival (80% vs 45%), none of the immunostaining algorithms was able to retain the prognostic impact of the groups (GCB vs non-GCB). In conclusion, stratification based on immunostaining algorithms should be used with caution in guiding therapy, even in clinical trials.

Beschreibung: Genomic studies have provided a complete profile of somatic mutations in chronic lymphocytic leukemia (CLL). These comprehensive approaches have revealed a relatively large number of mutated genes, the adverse prognostic value of some of which has been demonstrated in a number of reports. Recent studies have shown the clinical relevance of TP53 mutations at very low allele frequency. The presence and prognostic impact of minor mutated clones of other CLL driver genes and their clonal dynamics in the evolution of the disease is not well known. The goal of this study was to explore the presence of clonal and subclonal mutations of TP53, SF3B1, BIRC3, and ATM using an ultra-deep next-generation sequencing (NGS) strategy, to define the evolution of these subclones in different time-points of the disease, and to determine their influence in the outcome of the patients. Samples from 363 untreated CLL cases were included in this study. Copy number alterations were investigated by high density SNP-arrays or by quantitative PCR in 341 and 16 cases, respectively. Targeted ultra-deep NGS of TP53 (exons 4-10), ATM (exons 2-63), BIRC3 (exons 2-9), and SF3B1 (exons 14-16 and 18), including splicing sites, was performed using the Access-Array system (Fluidigm) and sequenced in a MiSeq equipment (Illumina). This methodology combined with a robust bioinformatic analysis based on well-known available tools allowed the identification of mutations down to 0.3% of variant allele frequency (VAF). Results obtained were fully verified by orthogonal techniques. Twelve per cent of VAF was used as threshold for the classification of clonal or subclonal mutations since 12% was the cut-off for detection of mutations by Sanger sequencing. Deletions of 11q comprising ATM or BIRC3 were found in 7% of the cases and were associated with mutations of the other ATM allele in 19/26 (73%) cases and BIRC3 in 3/23 (13%). Deletions of 17p were found in 19 (5%) cases and co-existed with TP53 mutations in 15 (79%) of them. Regarding the mutational status of the studied genes, TP53 mutations were present in 11.6% of patients (7.2% clonal, 4.4% subclonal), ATM mutations in 10% (7% clonal, 1% subclonal, 2% germline mutations considered pathogenic), SF3B1 mutations in 12% (7% clonal, 5% subclonal), and BIRC3 mutations in 4% (2% clonal, 2% subclonal). These subclonal mutations had similar molecular characteristics to their respective high-allele frequency mutations supporting a comparable pathogenic effect. In this regard, clonal and subclonal SF3B1 mutations were associated with shorter time to first treatment (TTT) independently of IGHV mutations. Clonal and subclonal TP53 mutations predicted for shorter overall survival (OS) together with the IGHV mutational status, although the impact of isolated TP53 mutations (i.e. without 17p deletion) on OS was not so evident, as has been the case in other studies. In addition, the outcome of patients with clonal and subclonal BIRC3 mutations showed a similar significant shorter OS. Regarding ATM, the effect of isolated subclonal ATM mutations could not be evaluated because of their low number, but ATM mutations as a whole had a significant impact on TTT even in the absence of 11q deletions. This study also reinforces the need to study the germline of the patients to fully characterize the ATM mutations observed in the tumors. Of note, germline variants previously described as pathogenic were associated with 11q deletions, confirming the hypothesis already suggested that these germline variants may influence disease progression through loss of the otherallele. Clonal dynamics was examined in longitudinal samples of 45 CLL patients. We confirmed the expansion of most TP53 mutated clones after therapy. However, both TP53 and SF3B1 mutations expanded also before any therapy in some patients, indicating that progressive dynamics of these clones is not only dependent on therapy selection. On the contrary, small ATM mutated clones seemed to be more stable. Although the number of cases is limited, we observed that clonal evolution in longitudinal samples had an unfavorable impact on OS. In conclusion, this study shows the presence of a high number of subclonal mutations of different driver genes in CLL and provides insights on the impact of these mutations on the outcome of the patients. These findings suggest that the characterization of the subclonal architecture may be relevant for a better management of CLL patients. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Introduction: DLBCL is biological and clinically highly heterogeneous. Although different genetic aberrations, including recurrent somatic mutations, have been described in this tumor, their clinical impact remains to be clarified. The aim of the present study was to determine somatic mutations and copy number alterations of a selected group of genes in patients with DLBCL, in order to assess their prognostic importance and to identify potential personalized targeted drugs for these patients. Methods: 150 patients (78M/72F; median age, 66 years) diagnosed with de novo DLBCL no otherwise specified at Hospital Clínic and other institutions of the GELCAB, treated with immunochemotherapy, were included in the study. An independent series of 111 patients (54M/57F; median age, 63 years), diagnosed at different Japanese and Spanish institutions, was used to validate the significant findings. Targeted next generation sequencing (NGS) of 106 representative genes related with DLBCL and Copy Number Alterations (CNA) assessment were performed. Ten functional pathways were pre-defined, including NOTCH, tumor suppressor genes, JAK/STAT, epigenome/chromatic modifier, BCR signaling, PI3K-AKT-mTOR, MAP-kinase, B-cell differentiation, immune surveillance and cell cycle alterations. Cell of origin (COO) of the tumors was established using gene expression or the Lymph2Cx assay. Genomic-guided potential therapeutic opportunities for each patient were identified in silico by a Cancer Genome Interpreter platform. Results: A total of 765 potential driver mutations were identified in 89 of the 106 genes with a slightly higher number in germinal center B-cell like (GCB) than activated B-cell-like (ABC) DLBCL subtype. The most frequently mutated genes found in 〉15% of the cases were KMT2D (MLL2), MYD88, CREBBP and TP53, with other 27 genes being mutated in 〉5% of the cases. Several genes were differentially mutated in GCB DLBCL subtype (KMT2D, CREBBP, TNFRSF14, B2M, EZH2, GNA13, FOXO1, ACTB and SOCS1) or ABC subtype (MYD88, PIM1, CD79B and PRDM1). No relevant differences were observed in the clinical features according to individual mutations or CNA. No single gene mutation predicted response to therapy. Genetic alterations in KLHL6, ETV6, SGK1, L8q12.1, CD79B, PIM1 and TP53 predicted poor OS, whereas mutations of SOCS1 were associated with better outcome. Alterations in NOTCH pathway and tumor suppressor pathway were associated with poor outcome, whereas those of JAK/STAT pathway showed favorable prognosis (see table for detailed data). NOTCH pathway (HR 2.8; p=0.006) and tumor suppressor pathway (HR 2.4; p=0.005) maintained independent significance for OS along with R-IPI (H 4.0; p=0.006) in a multivariate analysis that also included COO and beta2-microglobulin. In addition, the prognostic value of NOTCH and tumor suppressor pathways was confirmed in the independent validation series. Finally, we identified 69 cases (46%) carrying at least one genomic alteration in 9 genes considered a biomarker of drug response supported by data of early clinical trials or pre-clinical assays; tumors of additional 26 patients (17%) had at least one gene alteration that could be exploited by a drug repurposing strategy. Conclusions: Integrating the deep sequencing analysis of a panel of selected genes and CNA, we have recognized novel target genes and defined the clinical relevance of alterations of NOTCH and tumor suppressor pathways in DLBCL. Using an in silico prescription pipeline we have also identified a number of candidate drugs with potential therapeutic interactions with driver oncogenic proteins. All these findings may orient future preclinical and clinical intervention strategies in DLBCL. Table Initial features, response to therapy and outcome according to pathways´ status Table. Initial features, response to therapy and outcome according to pathways´ status Disclosures Sancho: Celltrion, Inc: Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Gonzalez Barca:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Speakers Bureau; Gilead: Speakers Bureau. Ohshima:Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; CHUGAI PHARMACEUTICAL CO.,LTD.: Research Funding, Speakers Bureau. Akashi:Sunitomo Dainippon Pharma: Consultancy; Celgene: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Bristol Meyers Squibb: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Shionogi & Co., Ltd: Research Funding; Astellas Pharma: Research Funding.

Beschreibung: Mantle cell lymphoma (MCL) is characterized by the translocation t(11;14)(q13;q32), which leads to the overexpression of cyclin D1. The cyclin D1-overexpression by itself is however not sufficient for lymphoma development. To identify other genes that are deregulated in MCL, we have performed integrated profiling of high-resolution array-based comparative genomic hybridization (array-CGH) with RNA-expression array analysis on the same set of MCL cases. DNA-alterations of MCL cases were assessed using a 3.6k BAC array with a resolution of 800 kb. On the same cases, RNA-expression array analysis was performed using the GeneChip® Human Genome U133 Plus 2.0 Array from Affymetrix. Integration of genomic-and transcription profiling data was performed using statistical tools. With array-CGH, we identified regions of chromosomal gain or loss and determined minimal common regions (mcr’s) by identifying the smallest region of overlap present in at least 36% of the cases. Integration of array-CGH and expression profiling was performed for genes located within the mcr’s and unsupervised clustering of gene-expression values within each mcr was performed. For several mcr’s, e.g. 1p22.1–31.1, 6q23.2–27 and 11q22.3–23.3, the clustering tree showed two groups, one with the chromosomal aberration and one without. Also, we observed that only a subset of genes located within a cytogenetic anomaly has a concomitant change in mRNA expression level. These genes are regulated by DNA-copy number (gene dosage). Amongst these, we identified several “hypothetical genes” and genes, which encode proteins involved in mitochondrial protein synthesis, the DNA damage repair pathway and the cAMP regulated pathway. This study shows the potential of the integrated profiling approach for identifying genes that are regulated by gene dosage (DNA-copy number). We anticipate that the genes we identified are important for MCL and it’s characteristic features like the low apoptosis rate and the chemotherapy-resistance.

Beschreibung: Acute myeloid leukemia with multilineage dysplasia (AML-MD) is recognized as a major AML category in the WHO classification, closely related to myelodysplasia and associated with poor prognosis. The biological background of this entity, however, has not been extensively assessed. In this context, we analyzed the gene expression profile in a series of patients with AML-MD from our institution. First, the transcriptional signature of diagnostic samples from 19 patients with AML-MD (age: 71, 30–93; 8M/11F) was analyzed, including 11 cases of intermediate-risk cytogenetics and 8 adverse karyotype samples. In a second step, we selected from the previous analysis those AML-MD patients with normal karyotype (NK AML-MD, n=8) and compared their genomic profile with 11 additional samples corresponding to normal karyotype AML without underlying multilineage dysplasia (NK AML). Overall gene expression was examined with oligonucleotide HGU133 Plus 2.0 arrays (Affymetrix), and gene expression measures were normalized using RMA methodology from the Affy package (Bioconductor project). Unsupervised two-dimensional cluster analysis of highly variable genes was performed with the dChip v1.3 software. In addition, supervised analysis to identify genes with significant differential expression was done with the Limma package (Bioconductor), which employs Bayesian statistics adjusted for multiple testing. Unsupervised analysis among AML-MD patients identified two major groups, mainly clustered according to cytogenetics: Group 1 (n=10) contained 9 patients with intermediate-risk cytogenetics whereas Group 2 (n=9) showed predominance of high-risk karyotype (78%; p=0.006). Supervised analysis allowed the identification of a cluster of 92 genes differentially expressed according to cytogenetics category, such as several ribosomal constituents and genes involved in translation (RPS20, EIF3S3, LOC400055), which were overexpressed in intermediate-risk AML-MD, or genes involved in the immune response (FCGR3A-3B, IL1R2, PLXNC1, FCAR, CLEC4D-4E, TNFRSF10C, C5R1), found to be highly expressed in AML-MD with high-risk cytogenetics. In a further analysis, gene expression profiling of NK AML-MD was compared to NK AML. Interestingly, unsupervised analysis revealed a clear distinction between both subgroups, with 1861 genes differentially expressed. Moreover, a subset of 34 genes, selected according to a high index of variable expression (log fold change〉1.0), showed a characteristic pattern in normal karyotype AML-MD. Among these, transcription factor TAL1, RNA-binding protein MSI2, and erythrocyte membrane protein RHAG were found to be overexpressed in NK AML-MD, while other genes such as RASSF4 and PIK3CD, involved in Ras signaling, or the transcription factor CEBPD were significantly underexpressed in this AML category. In conclusion, analysis of gene expression profile of AML-MD supports the biological heterogeneity of this AML category, partially associated with underlying cytogenetic abnormalities. Moreover, the striking distinctive profile observed in NK AML-MD suggests that multilineage dysplasia is associated to a specific transcriptional signature, distinguishable from normal cytogenetics AML lacking dysplastic features.

Beschreibung: Abstract 1236 Poster Board I-258 Glucocorticoids (GC) apoptotic effect in chronic lymphocytic leukemia (CLL) cells is known for many years. However, in CLL their use is often confined to their immunosuppressive activity in order to control autoimmune phenomena or as palliation. The fact that GC, particularly dexamethasone (DEX), can overcome p53 mediated resistance to therapy has renewed the interest in the use of GC as therapeutic agents in CLL. GC apoptotic induced cell death mechanism seems to depend on cell type and few studies were performed in CLL. GC increasing importance as apoptotic agent in CLL prompted us to analyze DEX apoptotic activity in CLL cells in order to clarify GC apoptotic mechanisms. For this, peripheral blood samples from 45 patients with CLL were selected for in vitro studies. Patients were analyzed for IGHV mutational status and ZAP-70 expression. Tumour cells were cultured over 24 hours with DEX at 13.25uM and viability was then determined by surface annexin V binding and propidium iodide (PI) staining flow cytometry analysis. To determine early apoptotic signal onset, BIM mRNA GC induced expression was quantified at different time points by quantitative RT-PCR. Genome-wide expression profile of CLL cells was done to discriminate genes involved in DEX apoptotic action. After 24 hours of exposure to therapeutic concentrations of DEX, cell viability was higher in mutated cases (M-CLL) than in unmutated IGHV cases (UM-CLL) (85.6% vs 69.5% in mean, respectively; p=0.000). mRNA BIM expression after 24 hours of treatment with DEX correlated with induced apoptotic cell death (R=0.496; p=0.000). As a consequence, UM-CLL had higher levels of induced mRNA levels of BIM than M-CLL cases (p=0.036). Time course experiments have shown that at 6h after DEX treatment BIM mRNA levels were induced 3 times without influence on cell viability. Genome-wide expression analysis of 12 CLL cases (7 UM-CLL, 5 M-CLL) was done after 6 hours of DEX treatment and was compared to the baseline gene expression. In both groups many genes were up and down regulated by DEX (UM-CLL 3359 genes and M-CLL 1008 genes, p adjusted 〈 0.01). Analysis of genes involved in the GC pathway revealed that basal mRNA levels of FKBP5, a protein essential to maintain the GC receptor (GR) complex suitable for GC binding, was more expressed in UM-CLL than in M-CLL cases (1.85 times, p= 0.027). Analysis by quantitative RT-PCR performed in 45 CLL patients to validate micro-array data confirmed that at baseline, FKPB5 expression was higher in UM-CLL than M-CLL (0.97 vs. 0.74 arbitrary units, respectively; p=0.042). The same was observed at protein level, WB analysis of FKBP5 basal levels showed that UM-CLL cases expressed more this protein than M-CLL (Fig. 1). In addition to that, genome-wide analysis revealed that GILZ, a glucocorticoid-induced leucine zipper, was differently induced by DEX in the studied groups. GILZ mRNA was less induced in M-CLL cases than in UM-CLL cases (difference fold change=0.52, p=0.0005). These results were also confirmed in 45 CLL cases by quantitative RT-PCR: in M-CLL, GILZ was induced 3.67 times whilst it was induced 5.62 times in UM-CLL cases (p=0.0001). In conclusion, treatment with GC induces more apoptotic cell death in UM-CLL than in M-CLL. As a downstream effect, BIM expression after GC exposure correlates with GC-induced apoptosis. Moreover, GC apoptotic effect in CLL is the result of several cell pathways imbalance as revealed by gene expression analysis. GILZ induction was proved to be necessary for DEX induced apoptosis in other cell types like multiple myeloma cell lines. In CLL, GILZ differential induction was observed at different degrees in GC-responders and non-responders. Finally, FKBP5 expression, upstream effecter of the GC pathway, correlated with cellular effect of GC and can be used to predict GC apoptotic activity in CLL cases. Figure 1 WB analysis of FKBP5 expression in CLL cells. Figure 1. WB analysis of FKBP5 expression in CLL cells. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Introduction: MCL is a mature B-cell neoplasm characterized by t(11;14) (q13;q32) and cyclin D1 (CCND1) overexpression. Molecular studies have revealed other alterations in cell-cycle regulation, DNA damage response and cell survival pathways, with a landscape of somatic mutations being recently identified. CNS involvement is a well known complication, occurring in 4-26% of MCL at five years, with an ominous significance. Although different clinical variables have been identified as risk factors for CNS infiltration, the biological parameters related to this complication have not been extensively studied. The aim of the study was to explore the biological parameters associated with CNS involvement in a multicentre and retrospective series of MCL patients. Patients and Methods: 285 patients (M:74%; 64 yr) diagnosed of MCL between 1990-2014 (median survival of 4 years) were analysed. In addition to standard clinico-biological variables, IGHV mutational analysis, chromosomal alteration studies and Sanger sequencing of NOTCH1, NOTCH2, TP53, BIRC3, WHSC1, MEF2B, MLL2, TLR2 and PRDM1 were performed. Results: CNS involvement was observed in 15/285 MCL patients (5.2%), with a 5-yr risk of 9.1% (95%CI: 4.6-13.6), one patient at diagnosis, and at first or second/ulterior progressions in 7 cases each. The clinical, pathological and molecular risk factors identified are detailed in the Table. In addition to what has been already described, CNS involvement was usually observed in MCL cases with a clinical nodal presentation (p=0.05). In fact, no indolent MCL with a non-nodal presentation developed this complication during the follow-up period. No differences were observed in the risk of CNS involvement between patients treated in first-line with conventional or high-dose intense treatment (5-yr risk: 6.1%+/-6% vs. 10.7%+/-10.6%, p=ns). Regarding the biological features, no differences in terms of the IGHV mutational status were observed in cases developing CNS involvement compared to the others (75% vs. 68.7%, using 97% identity cut-off). Similarly, the IGHV gene usage of CNS involved cases corresponded to the more frequent IGHV genes observed in MCL (usually IGHV1-18, IGHV3-23, IGHV4-34, IGHV4-59). Although not significant, a predominance of high number copy number alterations (CNA) (〉4) could be observed in the genetic study of MCL cases with CNS involvement as could be expected for the enrichment in blastoid variants (up to 50% of these cases). In fact, we did not observe any case with CNS involvement among those cases with 3 or less CNA. CNS involvement was not related to common poor prognosis genetic alterations such as 9p, 11p and 17p losses, but the presence of 8q gains was associated with a higher risk of CNS involvement (p=0.05). We did not find any significant association between CNS involvement and the large number of oncogenic mutations studied. Conclusions: CNS involvement in MCL is associated with initial aggressive clinico-biological characteristics. Non-nodal MCL cases with a low number of genetic alterations did not present CNS involvement. Finally, the presence of 8q gains was associated with a higher risk of CNS infiltration. Table Initial Clinical Features Category N 5 yr-CNS involvement (%, 95%CI) HR p Performance status (ECOG) 〉 1 8/51 41.5 (+/-28) 4.2 .003 ≤ 1 7/128 9.4 (+/-5.5) Nodal disease Yes 14/185 13.3 (+/-7.6) 6.1 .05 No 1/77 1.4 (+/-2.7) Hemoglobin (g/L) 〈 105 12/93 24.7 (+/-14.7) 3.2 .05 ≥ 105 3/78 5.3 (+/-6.3) LDH 〉 ULN 4/89 27.1(+/-19.4) 6.7 ULN 11/114 21.6 (+/-14.9) 3.5 .04 〈 ULN 3/66 8.7(+/-10) Molecular & Pathological data Histological variant Blastoid 6/58 17.3 (+/-13.7) 3.5 .02 Others 8/156 1.3 (+/-7.2) Ki-67 〉 30% 5/44 17.5 (+/-14.9) 3.6 .06 ≤ 30% 3/61 6.7 (+/-9.4) SOX11 Positive 8/153 2.9 (+/- 5.7) 2.6 ns Negative 1/42 2.1 (+/-2.4) IGHV ≥97% 6/109 9.5 (+/-9.6) 1.9 ns 4 2/87 3.9 (+/-5.7) 1.1 ns ≤ 4 1/44 2.3 (+/-4.3) Chromotripsis Yes 1/17 12.5 (+/-22) 3.2 ns No 2/106 1.9 (+/-2.7) 8q gain Yes 2/30 13.1(+/-19) 7.5 .05 No 2/97 1 (+/-1.96) CNA: copy number alteration; IGHV: immunoglobulin heavy chain; LDH: Lactate dehydrogenase Disclosures No relevant conflicts of interest to declare.