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We previously reported that t(14;18)-negative follicular lymphomas (FL) show a clear reduction of newly acquired N-glycosylation sites (NANGS) in immunoglobulin genes. We therefore aimed to investigate in-depth the occurrence of NANGS in a larger cohort of t(14;18)-positive and t(14;18)-negative FL, including early (I/II) and advanced (III/IV) stage treatment-naive and relapsed tumors. The clonotype was determined by using a next-generation sequencing approach in a series of 68 FL with fresh frozen material [36 t(14;18) positive and 32 t(14;18) negative]. The frequency of NANGS differed considerably between t(14;18)-positive and t(14;18)-negative FL stage III/IV, but no difference was observed among t(14;18)-positive and t(14;18)-negative FL stage I/II. The introduction of NANGS in all t(14;18)-negative clinical subgroups occurred significantly more often in the FR3 region. Moreover, t(14;18)-negative treatment-naive FL, specifically those with NANGS, showed a strong bias for IGHV4-34 usage compared with t(14;18)-positive treatment-naive cases with NANGS; IGHV4-34 usage was never recorded in relapsed FL. In conclusion, subgroups of t(14;18)-negative FL might use different mechanisms of B-cell receptor stimulation compared with the lectin-mediated binding described in t(14;18)-positive FL, including responsiveness to autoantigens as indicated by biased IGHV4-34 usage and strong NANGS enrichment in FR3.

We previously reported that t(14;18)-negative follicular lymphomas (FL) show a clear reduction of newly acquired N-glycosylation sites (NANGS) in immunoglobulin genes. We therefore aimed to investigate in-depth the occurrence of NANGS in a larger cohort of t(14;18)-positive and t(14;18)-negative FL, including early (I/II) and advanced (III/IV) stage treatment-naive and relapsed tumors. The clonotype was determined by using a next-generation sequencing approach in a series of 68 FL with fresh frozen material [36 t(14;18) positive and 32 t(14;18) negative]. The frequency of NANGS differed considerably between t(14;18)-positive and t(14;18)-negative FL stage III/IV, but no difference was observed among t(14;18)-positive and t(14;18)-negative FL stage I/II. The introduction of NANGS in all t(14;18)-negative clinical subgroups occurred significantly more often in the FR3 region. Moreover, t(14;18)-negative treatment-naive FL, specifically those with NANGS, showed a strong bias for IGHV4-34usage compared with t(14;18)-positive treatment-naive cases with NANGS; IGHV4-34usage was never recorded in relapsed FL. In conclusion, subgroups of t(14;18)-negative FL might use different mechanisms of B-cell receptor stimulation compared with the lectin-mediated binding described in t(14;18)-positive FL, including responsiveness to autoantigens as indicated by biased IGHV4-34usage and strong NANGS enrichment in FR3.

B-cell receptor (BCR) signals play a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL), but their role in regulating CLL cell proliferation has still not been firmly established. Unlike normal B cells, CLL cells do not proliferate in vitro upon engagement of the BCR, suggesting that CLL cell proliferation is regulated by other signals from the microenvironment, such as those provided by Toll-like receptors or T cells. Here, we report that BCR engagement of human and murine CLL cells induces several positive regulators of the cell cycle, but simultaneously induces the negative regulators CDKN1A, CDKN2A, and CDKN2B, which block cell-cycle progression. We further show that introduction of genetic lesions that downregulate these cell-cycle inhibitors, such as inactivating lesions in CDKN2A, CDKN2B, and the CDKN1A regulator TP53, leads to more aggressive disease in a murine in vivo CLL model and spontaneous proliferation in vitro that is BCR dependent but independent of costimulatory signals. Importantly, inactivating lesions in CDKN2A, CDKN2B, and TP53 frequently co-occur in Richter syndrome (RS), and BCR stimulation of human RS cells with such lesions is sufficient to induce proliferation. We also show that tumor cells with combined TP53 and CDKN2A/2B abnormalities remain sensitive to BCR-inhibitor treatment and are synergistically sensitive to the combination of a BCR and cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor both in vitro and in vivo. These data provide evidence that BCR signals are directly involved in driving CLL cell proliferation and reveal a novel mechanism of Richter transformation.

B-cell receptor (BCR) signals play a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL), but their role in regulating CLL cell proliferation has still not been firmly established. Unlike normal B cells, CLL cells do not proliferate in vitro upon engagement of the BCR, suggesting that CLL cell proliferation is regulated by other signals from the microenvironment, such as those provided by Toll-like receptors or T cells. Here, we report that BCR engagement of human and murine CLL cells induces several positive regulators of the cell cycle, but simultaneously induces the negative regulators CDKN1A, CDKN2A, and CDKN2B, which block cell-cycle progression. We further show that introduction of genetic lesions that downregulate these cell-cycle inhibitors, such as inactivating lesions in CDKN2A, CDKN2B, and the CDKN1A regulator TP53, leads to more aggressive disease in a murine in vivo CLL model and spontaneous proliferation in vitro that is BCR dependent but independent of costimulatory signals. Importantly, inactivating lesions in CDKN2A, CDKN2B, and TP53 frequently co-occur in Richter syndrome (RS), and BCR stimulation of human RS cells with such lesions is sufficient to induce proliferation. We also show that tumor cells with combined TP53 and CDKN2A/2B abnormalities remain sensitive to BCR-inhibitor treatment and are synergistically sensitive to the combination of a BCR and cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor both in vitro and in vivo. These data provide evidence that BCR signals are directly involved in driving CLL cell proliferation and reveal a novel mechanism of Richter transformation.

Splenic marginal zone lymphoma is a rare lymphoma. Loss of 7q31 and somatic mutations affecting the NOTCH2 and KLF2 genes are the commonest genomic aberrations. Epigenetic changes can be pharmacologically reverted; therefore, identification of groups of patients with specific epigenomic alterations might have therapeutic relevance. Here we integrated genome-wide DNA-promoter methylation profiling with gene expression profiling, and clinical and biological variables. An unsupervised clustering analysis of a test series of 98 samples identified 2 clusters with different degrees of promoter methylation. The cluster comprising samples with higher-promoter methylation (High-M) had a poorer overall survival compared with the lower (Low-M) cluster. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set of 36 patients. In the whole series, the High-M phenotype was associated with IGHV1-02 usage, mutations of NOTCH2 gene, 7q31-32 loss, and histologic transformation. In the High-M set, a number of tumor-suppressor genes were methylated and repressed. PRC2 subunit genes and several prosurvival lymphoma genes were unmethylated and overexpressed. A model based on the methylation of 3 genes (CACNB2, HTRA1, KLF4) identified a poorer-outcome patient subset. Exposure of splenic marginal zone lymphoma cell lines to a demethylating agent caused partial reversion of the High-M phenotype and inhibition of proliferation.

Splenic marginal zone lymphoma is a rare lymphoma. Loss of 7q31 and somatic mutations affecting the NOTCH2and KLF2genes are the commonest genomic aberrations. Epigenetic changes can be pharmacologically reverted; therefore, identification of groups of patients with specific epigenomic alterations might have therapeutic relevance. Here we integrated genome-wide DNA-promoter methylation profiling with gene expression profiling, and clinical and biological variables. An unsupervised clustering analysis of a test series of 98 samples identified 2 clusters with different degrees of promoter methylation. The cluster comprising samples with higher-promoter methylation (High-M) had a poorer overall survival compared with the lower (Low-M) cluster. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set of 36 patients. In the whole series, the High-M phenotype was associated with IGHV1-02 usage, mutations of NOTCH2gene, 7q31-32 loss, and histologic transformation. In the High-M set, a number of tumor-suppressor genes were methylated and repressed. PRC2 subunit genes and several prosurvival lymphoma genes were unmethylated and overexpressed. A model based on the methylation of 3 genes (CACNB2, HTRA1, KLF4)identified a poorer-outcome patient subset. Exposure of splenic marginal zone lymphoma cell lines to a demethylating agent caused partial reversion of the High-M phenotype and inhibition of proliferation.

Follicular lymphoma is characterized by aberrant BCL2 expression, a feature that is exploited for diagnostic purposes. However, a certain percentage of follicular lymphomas might be BCL2-negative by immunohistochemistry, increasing the difficulties in differentiating them from follicular hyperplasia. The expression of TUBB3 has been recently reported as negative in a small series of follicular lymphomas. We have therefore tested a larger series, including 61 BCL2-positive and 25 BCL2-negative cases, and compared them with 61 reactive lymphoid tissues. First, a subjective score of TUBB3 staining was applied, showing that it was consistently positive in reactive germinal centers, while most follicular lymphomas were negative; in fact, only 10/61 (16%) BCL2-positive and 1/25 (4%) BCL2-negative cases showed a positive staining for TUBB3, while 58/61 (95%) of tissues with follicular hyperplasia were positive. The application of a standardized scoring system to a large number of follicles, based on virtual slides, demonstrated that reactive lymphoid tissues had a significantly higher number of TUBB3-positive follicles both compared with BCL2-positive cases and to BCL2-negative cases. Our data support the use of TUBB3 staining in differentiating follicular lymphoma, including BCL2-negative cases, from follicular hyperplasia.

Follicular lymphoma is characterized by aberrant BCL2 expression, a feature that is exploited for diagnostic purposes. However, a certain percentage of follicular lymphomas might be BCL2-negative by immunohistochemistry, increasing the difficulties in differentiating them from follicular hyperplasia. The expression of TUBB3 has been recently reported as negative in a small series of follicular lymphomas. We have therefore tested a larger series, including 61 BCL2-positive and 25 BCL2-negative cases, and compared them with 61 reactive lymphoid tissues. First, a subjective score of TUBB3 staining was applied, showing that it was consistently positive in reactive germinal centers, while most follicular lymphomas were negative; in fact, only 10/61 (16%) BCL2-positive and 1/25 (4%) BCL2-negative cases showed a positive staining for TUBB3, while 58/61 (95%) of tissues with follicular hyperplasia were positive. The application of a standardized scoring system to a large number of follicles, based on virtual slides, demonstrated that reactive lymphoid tissues had a significantly higher number of TUBB3-positive follicles both compared with BCL2-positive cases and to BCL2-negative cases. Our data support the use of TUBB3 staining in differentiating follicular lymphoma, including BCL2-negative cases, from follicular hyperplasia.

Richter syndrome (RS) occurs in up to 15% of patients with chronic lymphocytic leukemia (CLL). Although RS, usually represented by the histologic transformation to a diffuse large B-cell lymphoma (DLBCL), is associated with a very poor outcome, especially when clonally related to the preexisting CLL, the mechanisms leading to RS have not been clarified. To better understand the pathogenesis of RS, we analyzed a series of cases including 59 RS, 28 CLL phase of RS, 315 CLL, and 127 de novo DLBCL. RS demonstrated a genomic complexity intermediate between CLL and DLBCL. Cell-cycle deregulation via inactivation of TP53 and of CDKN2A was a main mechanism in the histologic transformation from CLL phase, being present in approximately one half of the cases, and affected the outcome of the RS patients. A second major subgroup was characterized by the presence of trisomy 12 and comprised one third of the cases. Although RS shared some of the lesions seen in de novo DLBCL, its genomic profile was clearly separate. The CLL phase preceding RS had not a generalized increase in genomic complexity compared with untransformed CLL, but it presented clear differences in the frequency of specific genetic lesions.

B cell receptor (BCR) signals play a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL), but their role in regulating CLL cell proliferation has still not been firmly established. Unlike normal B cells, CLL cells do not proliferate in vitroupon engagement of the BCR, suggesting that CLL cell proliferation is regulated by other signals from the microenvironment, such as those provided by Toll-like receptor (TLR) ligands or T cells. However, the rapid reduction in the percentage of proliferating CLL cells in patients treated with a BTK or SYK inhibitor suggests that the BCR may be more directly involved, at least in a subset of cases.

Anaplastic large-cell lymphomas (ALCLs) are a group of clinically and biologically heterogeneous diseases including the ALK+ and ALK− systemic forms. Whereas ALK+ ALCLs are molecularly characterized and can be readily diagnosed, specific immunophenotypic or genetic features to define ALK− ALCL are missing, and their distinction from other T-cell non-Hodgkin lymphomas (T-NHLs) remains controversial. In the present study, we undertook a transcriptional profiling meta-analysis of 309 cases, including ALCL and other primary T-NHL samples. Pathway discovery and prediction analyses defined a minimum set of genes capable of recognizing ALK− ALCL. Application of quantitative RT-PCR in independent datasets from cryopreserved and formalin-fixed paraffin-embedded samples validated a 3-gene model (TNFRSF8, BATF3, and TMOD1) able to successfully separate ALK− ALCL from peripheral T-cell lymphoma not otherwise specified, with overall accuracy near 97%. In conclusion, our data justify the possibility of translating quantitative RT-PCR protocols to routine clinical settings as a new approach to objectively dissect T-NHL and to select more appropriate therapeutic protocols.

Anaplastic large-cell lymphomas (ALCLs) are a group of clinically and biologically heterogeneous diseases including the ALK+and ALK−systemic forms. Whereas ALK+ALCLs are molecularly characterized and can be readily diagnosed, specific immunophenotypic or genetic features to define ALK−ALCL are missing, and their distinction from other T-cell non-Hodgkin lymphomas (T-NHLs) remains controversial. In the present study, we undertook a transcriptional profiling meta-analysis of 309 cases, including ALCL and other primary T-NHL samples. Pathway discovery and prediction analyses defined a minimum set of genes capable of recognizing ALK−ALCL. Application of quantitative RT-PCR in independent datasets from cryopreserved and formalin-fixed paraffin-embedded samples validated a 3-gene model (TNFRSF8, BATF3, and TMOD1) able to successfully separate ALK−ALCL from peripheral T-cell lymphoma not otherwise specified, with overall accuracy near 97%. In conclusion, our data justify the possibility of translating quantitative RT-PCR protocols to routine clinical settings as a new approach to objectively dissect T-NHL and to select more appropriate therapeutic protocols.

The BIOMED-2 protocol is widely used for detecting clonality in lymphoproliferative disorders. The protocol requires multiple PCR reactions, which are analyzed by either capillary electrophoresis (GeneScan analysis) or heteroduplex PAGE analysis. We tested a microfluidic chip-based electrophoresis device (Agilent 2100 Bioanalyzer) for the analysis of B-cell clonality using PCR for the three framework subregions (FR) of the Ig heavy chain gene (IGH) and PCR for two rearrangements occurring in the Ig κ chain gene (IGK-VJ and IGK-DE). We analyzed 62 B-cell lymphomas (33 follicular and 29 nonfollicular) and 16 reactive lymph nodes. Chip-based electrophoresis was conclusive for monoclonality in 59/62 samples; for 20 samples, it was compared with GeneScan analysis. Concordant results were obtained in 45/55 IGH(FR1, FR2, and FR3) gene rearrangements, and in 34/37 IGKgene rearrangements. However, when the chip device was used to analyze selected IGKgene rearrangements (biallelic IGKrearrangements or IGKrearrangements in a polyclonal background), its performance was not completely accurate. We conclude, therefore, that this microfluidic chip-based electrophoresis device is reliable for testing cases with dominant PCR products but is less sensitive than GeneScan in detecting clonal peaks in a polyclonal background for IGHPCR, or with complex IGKrearrangement patterns.

S100A1 is a calcium-binding protein, which has been recently found in renal cell neoplasms. We evaluated the diagnostic utility of immunohistochemical detection of S100A1 in 164 renal cell neoplasms. Forty-one clear cell, 32 papillary, and 51 chromophobe renal cell carcinomas, and 40 oncocytomas, 164 samples of normal renal parenchyma adjacent to the tumors and 13 fetal kidneys were analyzed. The levels of S100A1 mRNA detected by quantitative RT-PCR analysis of frozen tissues from seven clear cell, five papillary, and six chromophobe renal cell carcinomas, four oncocytomas, and nine samples of normal renal tissues adjacent to neoplasms were compared with the immunohistochemical detection of protein expression. Clear cell and papillary renal cell carcinomas showed positive reactions for S100A1 in 30 out of 41 tumors (73%) and in 30 out of 32 (94%) tumors, respectively. Thirty-seven renal oncocytomas out of 40 (93%) were positive for S100A1, whereas 48 of 51 (94%) chromophobe renal cell carcinomas were negative. S100A1 protein was detected in all samples of unaffected and fetal kidneys. S100A1 mRNA was detected by RT-PCR in all normal kidneys and renal cell neoplasms, although at very different levels. Statistical analyses comparing the different expression of S100A1 in clear cell and chromophobe renal cell carcinomas observed by immunohistochemical and RT-PCR methods showed significant values (P<0.001), such as when comparing by both techniques the different levels of S100A1 expression in chromophobe renal cell carcinomas and oncocytomas (P<0.001). Our study shows that S100A1 protein is expressed in oncocytomas, clear cell and papillary renal cell carcinomas but not in chromophobe renal cell carcinomas. Its immunodetection is potentially useful for the differential diagnosis between chromophobe renal cell carcinoma and oncocytoma. Further, S100A1 protein expression is constantly detected in the normal parenchyma of the adult and fetal kidney.Modern Pathology (2007) 20, 722–728; doi:10.1038/modpathol.3800828; published online 4 May 2007

S100A1 is a calcium-binding protein, which has been recently found in renal cell neoplasms. We evaluated the diagnostic utility of immunohistochemical detection of S100A1 in 164 renal cell neoplasms. Forty-one clear cell, 32 papillary, and 51 chromophobe renal cell carcinomas, and 40 oncocytomas, 164 samples of normal renal parenchyma adjacent to the tumors and 13 fetal kidneys were analyzed. The levels of S100A1 mRNA detected by quantitative RT-PCR analysis of frozen tissues from seven clear cell, five papillary, and six chromophobe renal cell carcinomas, four oncocytomas, and nine samples of normal renal tissues adjacent to neoplasms were compared with the immunohistochemical detection of protein expression. Clear cell and papillary renal cell carcinomas showed positive reactions for S100A1 in 30 out of 41 tumors (73%) and in 30 out of 32 (94%) tumors, respectively. Thirty-seven renal oncocytomas out of 40 (93%) were positive for S100A1, whereas 48 of 51 (94%) chromophobe renal cell carcinomas were negative. S100A1 protein was detected in all samples of unaffected and fetal kidneys. S100A1 mRNA was detected by RT-PCR in all normal kidneys and renal cell neoplasms, although at very different levels. Statistical analyses comparing the different expression of S100A1 in clear cell and chromophobe renal cell carcinomas observed by immunohistochemical and RT-PCR methods showed significant values (P<0.001), such as when comparing by both techniques the different levels of S100A1 expression in chromophobe renal cell carcinomas and oncocytomas (P<0.001). Our study shows that S100A1 protein is expressed in oncocytomas, clear cell and papillary renal cell carcinomas but not in chromophobe renal cell carcinomas. Its immunodetection is potentially useful for the differential diagnosis between chromophobe renal cell carcinoma and oncocytoma. Further, S100A1 protein expression is constantly detected in the normal parenchyma of the adult and fetal kidney.

Gastrointestinal stromal tumors (GISTs) frequently harbor mutations in the KITand PDGFRAgenes, the presence and type of which correlate with the response to the kinase inhibitor imatinib mesylate. Because most GIST mutations are deletions/insertions, we used a microfluidic apparatus to detect these size variations in polymerase chain reaction-amplified DNA. This approach, termed microfluidic deletion/insertion analysis (MIDIA), identified mutations in 30 of 50 DNA samples from paraffin-embedded CD117-positive GISTs (60%), comprising 25 deletions and five insertions. Sequencing of 14 MIDIA-positive samples confirmed the deletions/insertions, including two 3-bp alterations. Sequencing of all 20 MIDIA-negative samples also showed highly consistent results with MIDIA because 10 cases were wild type and eight displayed a single base substitution in which detection by MIDIA was not expected. Sequencing also revealed a 3-bp deletion undetected by MIDIA, thus establishing the resolution limit of MIDIA at deletions/insertions ≥3 bp. Denaturing high-pressure liquid chromatography analysis confirmed all mutations detected by MIDIA and sequencing. We pro-pose MIDIA as the first step in mutational screening of GIST because it allowed the detection of 75% of mutated cases (94% of deletions/insertions) in less than 30 minutes after polymerase chain reaction amplification and at a lower cost compared with denaturing high-pressure liquid chromatography and sequencing, which might then be used only for MIDIA-negative cases.

Composite lymphomas are rare associations of two distinct lymphoma types at the same anatomical site. Reporting of such cases is important because they pose major biologic, diagnostic, and therapeutic dilemmas. In this study, we describe the third reported case of mantle cell and follicular lymphoma. We performed accurate immunohistochemical and molecular studies to define the mono- vs biclonal nature of this neoplasm. We used both manual and LASER-capture microdissection combined to multiple molecular approaches for clonality determination, including detection of heavy and light chain recombination, as well as presence of κ/Kde recombination and sequence analysis. By immunohistochemistry and FISH, we confirmed the presence of two distinct lymphoma types characterized by specific translocations [namely, t(11;14) and t(14;18)], while we demonstrated two distinct and not clonally related cell populations by molecular techniques. The light chain approach, and particularly the kappa and κ/Kde recombination detection, proved very useful for solving the clonality issue.Composite lymphomas are rare associations of two distinct lymphoma types at the same anatomical site. Reporting of such cases is important because they pose major biologic, diagnostic, and therapeutic dilemmas. In this study, we describe the third reported case of mantle cell and follicular lymphoma. We performed accurate immunohistochemical and molecular studies to define the mono- vs biclonal nature of this neoplasm. We used both manual and LASER-capture microdissection combined to multiple molecular approaches for clonality determination, including detection of heavy and light chain recombination, as well as presence of κ/Kde recombination and sequence analysis. By immunohistochemistry and FISH, we confirmed the presence of two distinct lymphoma types characterized by specific translocations [namely, t(11;14) and t(14;18)], while we demonstrated two distinct and not clonally related cell populations by molecular techniques. The light chain approach, and particularly the kappa and κ/Kde recombination detection, proved very useful for solving the clonality issue.

p63, a member of the p53 family, is involved in the survival and differentiation of reserve/stem cells in different epithelia. To unveil the possible role of p63 in thymic physiology and pathology, we investigated the expression of p63 isoforms in normal thymus, thymomas and other mediastinal tumours. All samples were analysed using immunohistochemistry with three different antibodies: 4A4 antibody recognising all p63 isoforms, p40 antibody reacting only with truncated dominant-negative isoforms (?N-p63) and H-129 antibody recognising all alpha-isoforms. Reverse-transcription polymerase chain reaction (RT-PCR), and real-time PCR analyses were performed on RNA extracted from frozen samples of four thymomas and two primary-mediastinal large-B-cell lymphoma (PMLBCL). In normal thymus, ?N-p63a was expressed in all cortical and medullary epithelial cells, with decreasing intensity in Hassall's corpuscles. This phenotype was conserved in neoplastic transformation since all 54 investigated thymomas (World Health Organization types A, AB, B1, B2, B3, C) expressed ?N-p63a (virtually 100% cells). The predominance of ?N-p63a isoform mRNA was confirmed by real-time PCR. Among other mediastinal tumours, ?N-p63a was only expressed in those displaying either a stratified epithelial component (teratomas) or epidermoid differentiation (lung carcinoma). Among lymphomas, T-cell-precursor lymphomas did not express p63, whereas most PMLBCL expressed TA-p63a (7/8).

To investigate the molecular events that may underpin dysfunctional repair processes that characterize idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), we analyzed the expression patterns of β-catenin on 20 IPF/UIP lung samples, together with two downstream target genes of Wnt signaling, cyclin-D1, and matrilysin. In 18 of 20 cases of IPF/UIP investigated on serial sections, nuclear β-catenin immunoreactivity and abnormal levels of cyclin-D1 and matrilysin were demonstrated in proliferative bronchiolar lesions (basal-cell hyperplasia, squamous metaplasia, bronchiolization, honeycombing). The nature of these lesions was precisely defined using specific markers (ΔN-p63, surfactant-protein-A, cytokeratin-5). Interestingly, nuclear β-catenin accumulation was also demonstrated in fibroblast foci in most (16 of 20) IPF/UIP samples, often associated with bronchiolar lesions. Similar features were not observed in normal lung and other fibrosing pulmonary diseases (diffuse alveolar damage, organizing pneumonia, nonspecific interstitial pneumonia, desquamative interstitial pneumonia). Sequence analysis performed on DNA extracted from three samples of IPF/UIP did not reveal abnormalities affecting the β-catenin gene. On the basis of these findings new models for IPF/UIP pathogenesis can be hypothesized, centered on the aberrant activation of Wnt/β-catenin signaling, with eventual triggering of divergent epithelial regeneration at bronchiolo-alveolar junctions and epithelial-mesenchymal-transitions, leading to severe and irreversible remodeling of the pulmonary tissue.

The expression of BCL6 in B cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase involved in the degradation of BCL6 and is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11mutations are found in 23% of Burkitt lymphoma (BL) patients. FBXO11mutations impaired BCL6 degradation and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of either one or two copies of the FBXO11gene in mice cooperated with oncogenic MYC and accelerated B cell lymphoma onset, providing experimental evidence that FBXO11 is a haplo-insufficient oncosuppressor in B cell lymphoma. In WT and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degrader or inhibitors partially impaired lymphoma growth in vitroand in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11mutations in BL we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches such as BCL6 degraders and direct MYC inhibition could be exploited as a targeted therapy for BL.

Ladetto: Roche: Honoraria; Celgene: Honoraria; Acerta: Honoraria; Jannsen: Honoraria; Abbvie: Honoraria; Sandoz: Honoraria. Di Rocco:Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Rossi:Novartis: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Janssen: Membership on an entity's Board of Directors or advisory committees, Travel expenses; Roche: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Mundipharma: Honoraria; Sandoz: Honoraria; Seattle Genetics: Research Funding; Alexion: Other: Travel expenses. Chiappella:Janssen: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Roche: Other: lecture fees; Teva: Other: lecture fees; Nanostring: Other: lecture fees; Amgen: Other: lecture fees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees. Rusconi:Celgene: Research Funding. Gomes da Silva:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Institution's payment for consultancy, Travelling support; Celgene: Other: Travelling support; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Roche: Other: Institution's payment for consultancy, Travelling support; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Research Funding. Vitolo:Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Speakers Bureau. Martelli:Sandoz: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; F. Hoffman-La Roche: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background.Ara-c based chemo-immunotherapy followed by autologous stem cell transplantation (ASCT) is the most effective approach in young mantle cell lymphoma (MCL) patients, though few if any patients are cured. Recent data indicate that subsequent Rituximab maintenance (RM) prolongs PFS and OS (Le Gouill NEJM 2017). Lenalidomide is an oral agent effective in MCL, considered suitable for prolonged maintenance programs, but has never been tested in this setting. The FIL MCL0208 trial (NCT02354313) is a prospective, international randomized, phase III trial, comparing Lenalidomide maintenance (LM) vs observation (OBS) after an intensive Ara-c containing chemo-immunotherapy (R-HDS) program, followed by ASCT in previously untreated MCL patients.

INTRODUCTION

Background and aims.Data collection in clinical trials is becoming complex, with huge amount of clinical and biological variables that need to be recorded, verified and analyzed to confirm obvious and identify unexpected associations. Outside the medical field, data warehouses (DWs) are widely employed to achieve these objectives. To verify whether DWs might be useful for data quality and association analysis, a team of clinicians, biologists, statisticians and biomedical engineers developed DELPHI (“Data ELaboration to Predict Hypothetical assocIations”). The tool has been suited to a large clinical trial from the Fondazione Italiana Linfomi (FIL), including all the clinical, biological and mutational features collected both in the trial and in the ancillary studies. Herein we present the first results, related to baseline variables: 1) creation of a broad DW (including 350 variables for 300 patients, thus > 105data); 2) data harmonization and quality control; 3) discovery of unexpected associations and (in a later phase) outcome correlations; 4) results visualization.

Luminari: Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees.

No relevant conflicts of interest to declare.

INTRODUCTION:We recently described inactivating mutations of the FBXO11 gene in Diffuse Large B Cell Lymphoma (DLBCL). One major function of FBXO11 is to regulate BCL6 stability as well as other targets such as SNAIL. BCL6 acts as an oncogene in several human B-cell lymphomas and its expression levels can be increased by several mechanisms including chromosomal translocations, point mutations in the promoter region and reduced degradation by inactivation of FBXO11. Thus, FBXO11 acts as an oncosuppressor in DLBCL by promoting the accumulation of BCL6. However, a clear and complete picture of the distribution of FBXO11 mutations in different lymphoma subtypes is missing, and the in vivofunctions of FBXO11 in normal tissue and lymphoma development are completely lacking. In the present work, we investigated the frequency and distribution of FBXO11 mutations in an extended panel of human BCL6 positive lymphoma and we functionally validated novel FBXO11 mutations for their ability to induce BCL6 degradation.

Background.The aggressive clinical behavior of mantle cell lymphoma (MCL) is attributed to specific genetic and molecular mechanisms involved in its pathogenesis, mainly the t(11;14)(q13;q32) traslocation and cyclin D1 (CCND1) overexpression. Nevertheless, evidence of a certain degree of clinical/biological heterogeneity has been disclosed by gene expression profile (GEP) and (immuno)genetic/immunohistochemistry studies.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Tumor necrosis factor (TNF)-like cytokine 1A (TL1A) is a member of the TNF superfamily, expressed on dendritic cells, macrophages, lymphocytes, and endothelial cells. It is the only described ligand for death receptor 3 (DR3), a death-domain containing receptor of the TNF-receptor superfamily, mainly expressed on lymphocytes, natural killer (NK) cells, and NK-T cells. TL1A–DR3 interaction results in co-stimulatory signaling for activated T cells, leading to amplification of inflammation and immune responses, correlated with greater pathogenicity in diverse autoimmune diseases. In contrast, in activated B cells the TL1A/DR3 system exerts inhibitory effect on cell proliferation, suggesting that TL1A may also have modulatory and homeostatic functions on B-cell expansion. Chronic lymphocytic leukemia (CLL) is the leukemia with the highest incidence among adults in Western countries. It is well documented that several elements within the tumor microenvironment, including antigens, cytokines, adhesion molecules, and surface receptors, play a fundamental role in supporting the growth of CLL. In contrast, little is known on regulatory mechanisms of CLL growth. In this study, we have investigated the possible regulatory role of the TL1A/DR3 system in B cells from CLL patients.

Background:stabilizing mutations of NOTCH1have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV(IGHV-UM), immuno-chemorefractory or advanced disease phase CLL, and have been associated with particularly unfavourable prognosis (Rossi et al, Blood, 2012; Del Poeta et al, Br J Haematol, 2013; Stingelbauer et al, Blood, 2014). In CLL, all NOTCH1mutations disrupt the C-terminal PEST domain (about 80% of which are a 7544-7545delCT frameshift deletion) and cause an accumulation of an active NOTCH1 isoform.

A test series of 101 samples, including 3 SMZL cell lines (Karpas1718, VL51, SSK41) and 98 clinical SMZL cases from 10 Centers and, a validation set of 36 SMZL cases from 2 additional Centers were analyzed with the Illumina Infinium HumanMethylation27 arrays, as described (BJH 2013). Gene GEP was available for a subset of 11/101 cases. Methylation profiles were correlated with the presence of the copy number aberrations (CNAs) and somatic mutations recurrent in SMZL, IGHV mutational status, HCV infection and clinical data. Probes mapped outside CpG islands were discarded. Supervised analyses were performed using a t-test on the continuous beta values, followed by multiple test correction. For Fisher’s test, probes were classified into unmethylated (beta-values <0.3) or methylated (beta-value >0.3).Unsupervised clustering analysis of 101 SMZL samples identified 2 clusters.Cluster one (23/101, 23%) comprised the 3 cell lines and included cases characterized by a generalized higher degree of promoter methylation (High-M), and had a significantly poorer overall survival (OS) than the remaining 79/101 (77%) cases (Low-M) (P=0.048; HR 2.4; 95% CI, 1-5.8).To better understand the biology underlying this clustering, supervised analysis comparing High-M to Low-M cases was performed. By combining t- and Fisher's tests, 3200 gene probes showed differential methylation status between High-M and Low-M lymphomas. This probes signature was significantly enriched of promoters of promoter regions of PRC2-complex targets, genes involved in chromatin remodeling, and HOX and SOX family genes, and genes down-regulated by CDH1. Genes coding for subunits of the PRC2-complex (EZH2, EED, SUZ12) appeared unmethylated in their promoter regions and over-expressed by GEP in the High-M set, while EZH2-target genes and genes harboring the H3K27me3 mark had methylated promoters and down-regulated expression. A number of reported tumor-suppressor genes were highly methylated in High-M cases, including KLF4, DAPK1, CDKN2A/B, CDKN1C, CDH1, CDH2 and TIMP3. Known pro-survival lymphoma genes, such as SYK, MCL1, CARD11, BIRC5, BTK, BCL10, FOXP1, CD40, TACI and TCL1A/B, were un-methylated and over-expressed in High-M cases.The prognostic relevance of the High-M phenotype was confirmed in an independent validation set. An unsupervised clustering using the top-100 differentially methylated probes obtained by t-test in the test series identified two clusters with one (11/36, 30%) bearing methylation profile similar to High-M and a poorer OS (P=0.038).All the samples were then pooled to improve the statistical power of further analyses regarding the impact of methylation on the clinical course and on the association with biologic variables. High-M was significantly associated with poorer OS (P=0.008; HR 2.58; 95% CI, 1.2-5.4), transformation into aggressive lymphoma (33% vs 0.02%, P<0.001) and with 7q31-33 loss (38% vs 16%, P=0.026). In addition, High-M showed a higher frequency of NOTCH-pathway somatic mutations (39% vs 16%, P=0.063). HCV was more frequent in Low-M (20% vs 4%, P=0.058).In a Cox regression model adjusted for the variables significantly associated with outcome at univariate analysis [High-M, age>60, Interguppo Italiano Linfomi (IIL) SMZL score, HCV infection], High-M (HR 16; 95% CI, 1.3-201) and IIL SMZL score (HR 4.5; 95% CI, 1.3-15) retained their prognostic significance on OS (P<0.001).Genome-wide promoter-methylation profiling in splenic MZL identified a subset of patients with poorer OS, characterized by a high promoter methylation and by specific genetic and biologic features. These data may provide the rational to explore epigenetic drugs in SMZL. *, AJA and AR equally contributed.No relevant conflicts of interest to declare.

SMZL usually has an indolent clinical course. Transformation to aggressive lymphoma occurs in only 4-5% of cases. Tools to identify patients facing a more aggressive clinical course and targeted therapies are needed. In the last years, we have characterized SMZL by DNA profiling (Blood 2011) and RNA/miRNA expression profiling (Mod Path 2013), and shown that somatic mutations of NOTCH2 and other genes involved in the pathway are frequent and mutually exclusive with mutations affecting NKFB pathway (JEM 2012). Here, we report genome-wide DNA promoter methylation profiling of 137 SMZL samples with its clinical and biologic implications.

No relevant conflicts of interest to declare.

activating mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM) CLL, chemorefractory CLL and CLL in advanced disease phases. In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain and cause an accumulation of an active NOTCH1 isoform. Notably, about 80% of NOTCH1 mutations are represented by a CT frameshift deletion at nucleotides 7544–7545 (c.7544–7545delCT). Clinically, the presence of NOTCH1 mutations is an independent predictor of overall survival in CLL and identifies a subset of patients with particularly unfavourable prognosis (Rossi et al, Blood, 119, 521, 2012).to identify peculiar molecular and biological features of NOTCH1 mutated CLL in the context of IGHV-UM CLL.the presence of the c.7544–7545delCT NOTCH1 frameshift deletion was investigated by an ad-hoc amplification refractory mutation system (ARMS) PCR set up to obtain an amplicon specific for the NOTCH1 mutated form and a second amplicon as control. The percentage of NOTCH1 DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR), calculating the ratio between the amount of the specific NOTCH1 mutated amplicon and the amount of the control amplicon, the latter representing the total amount of NOTCH1 DNA irrespective of its mutational status. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4×44K Agilent platform. The differential expression of specific genes/proteins was validated by QRT-PCR, western blotting and immunohistochemistry. A BrdU uptake assay was used to evaluate proliferation of CLL cells by CpG/IL2 stimulation.in a cohort of 380 IGHV-UM CLL, the c.7544–7545delCT NOTCH1 mutation was found in 83/380 (21.8%) cases. QRT-PCR revealed a percentage of NOTCH1 mutated DNA ranging from 1 to 37%. CLL cases carrying the c.7544–7545delCT NOTCH1 mutation (NOTCH1-Mut) showed higher NOTCH1 protein expression than CLL cases lacking NOTCH1-Mut employing monoclonal antibodies either recognizing the trans-membrane (mean fold increase=3) or the intra-citoplasmic (mean fold increase=2.1) NOTCH1 domain. A GEP comparing RNA from purified CLL samples of 5 NOTCH1-Mut CLL and 5 CLL lacking NOTCH1-Mut was performed, selecting the 5 NOTCH1-Mut cases among those with the higher percentages of NOTCH1 mutated DNA (percentages of NOTCH1 mutated DNA ranging from 15 to 37%). This approach selected the nucleophosmin 1 gene (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-Mut CLL cases. A higher expression of the above mentioned genes in NOTCH1-Mut CLL was validated in a wider series of 34 cases (18 NOTCH1-Mut cases; NPM1, p=0.03; RPS6, p=0.045; RPS10, p=0.048; RPS17, p=0.048; RPS28, p=0.049; RPSA, p=0.048; RPL7A, p=0.039; RPL18, p=0.041, respectively). Western blot analysis in 8 cases (4 NOTCH1-Mut cases) confirmed a higher NPM1 expression in NOTCH1-Mut cases (range of fold increase from 1.6 to 5.2) also at protein level. Consistently, lymph nodes preparations from NOTCH1-Mut CLL cases revealed a strong NPM1 staining both in nucleoli and cytoplasms. Finally, when stimulated in-vitro with the CpG/IL2 combination, NOTCH1-mut IGHV-UM CLL cells proliferated, as detected by a BrdU uptake assay (>10 fold increase over control), and up-regulated NPM1 both at transcript (mean fold increase=2.02 after 18 hours of CpG exposure, p=0.001) and protein (fold increase of 1.34 after 6 hours of CpG exposure) levels.NPM1 was identified as constitutively overexpressed in NOTCH1-Mut IGHV-UM CLL together with several ribosome-associated components. These findings are suggestive for an increased activity of the ribosomal machinery in NOTCH1-Mut IGHV-UM CLL as part of the molecular processes leading to control of CLL cell growth and survival in this clinically unfavourable disease subset.No relevant conflicts of interest to declare.

Abstract 3880

Abstract 1772

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Diffuse large B cell lymphoma (DLBCL) represents a group of aggressive lymphoid neoplasms with morphological variants. Plasmablastic lymphoma has been recently classified as a clinical-pathological variant with plasmablastic features and terminal B-cell differentiation. Diffuse large B-cell lymphoma with both plasmablastic and plasmacytic features has been previously reported, but cell lines and animal models have been never described. However, the use of human lymphoma cell lines and animal models contributes significantly to a better understanding of the pathophysiology of hematopoietic tumors.We established and characterized a new plasmablastic lymphoma cell line with plasmacytic differentiation and we tested its tumorigenicity in vivo.Mononuclear cells from human DLBCL bone marrow, were seeded at concentration of 1×106/mL in RPMI 10% FBS medium added with 1% of antibiotics. Cells showed spontaneous proliferative capacity and a cell line (VR09) was established after a few months of continuous culture. VR09 cells were evaluated by May-Grünwald-Giemsa staining, flow cytometry, immunohistochemistry, molecular biology, standard karyotyping and fluorescence in situ hybridization (FISH). Epstein-Barr Virus (EBV) positivity was also tested. Moreover, cells were inoculated subcutaneously into 6 immunodeficient Rag2−/−γ-chain−/− mice. Subcutaneously-growing tumors were evaluated by immunohistochemistry and FISH. Disaggregated cells from masses were cultured again to confirm their biological features and proliferative capacity.Cells in suspension formed large clumps with round shape by ten weeks of continuous culture. Cells appeared morphologically as medium/large-size cells with plasmablastic/plasmacytic appearance and, sometimes, bizarre shape. They had a round-ovoid or irregular nucleus with compact chromatin, no nucleoli and an abundant basophilic cytoplasm.When subcutaneously injected in mice, VR09 cells grew as an isolated spherical tumor (100% incidence). Moreover, cells maintained their proliferative capacity once disaggregated from mass and re-cultured.Cells displayed the phenotype of plasmablastic lymphoma with secretory differentiation (CD19+ CD3- CD5- CD20+ CD79a+ CD79b+/− CD138+ CD38+ cycline D1- Ki6780% IgM+ IgD+ MUM1+ MDNA+ CD10- CD22+ CD23+ CD43+ K+, λ- Bcl2+ Bcl6-). The same markers were detected on tissues derived from growing tumors and cell suspension.FISH analysis revealed the trisomy of chromosome 12 and the negativity for the MYC rearrangement both in VR09 cell line and in tumors. Cell suspension from mass showed an additional structural chromosome aberration involving chromosomes 7 and 9.EBER probe detected episomal EBV genome in both VR09 cell line and tumors.Genetic analysis in VR09 cell line showed the somatic hypermutation in the VH region, wild type p53 and a novel synonymous mutation of CD79B-variant 2, involving the insertion of alanine in the exon 2.We have established and characterized a new plasmablastic EBV-positive cell line with focal plasmacytic differentiation and with tumorigenic potential in immunodeficient mice. VR09 cell line represents a model for in vitro and in vivo studies regarding pathogenesis and drug sensitivity.No relevant conflicts of interest to declare.

Abstract 263

Abstract 5163

Abstract 2668