Alberto Arribas

Nodal marginal zone lymphoma: gene expression and miRNA profiling identify diagnostic markers and potential therapeutic targets

Nodal marginal zone lymphoma (NMZL) is a small B-cell neoplasm whose molecular pathogenesis is still essentially unknown and whose differentiation from other small B-cell lymphomas is hampered by the lack of specific markers. We have analyzed gene expression, miRNA profile, and copy number data from 15 NMZL cases. For comparison, 16 follicular lymphomas (FLs), 9 extranodal marginal zone lymphomas, and 8 reactive lymph nodes and B-cell subtypes were included. The results were validated by quantitative RT-PCR in an independent series, including 61 paraffin-embedded NMZLs. NMZL signature showed an enriched expression of gene sets identifying interleukins, integrins, CD40, PI3K, NF-κB, and TGF-β, and included genes expressed by normal marginal zone cells and memory B cells. The most highly overexpressed genes were SYK, TACI, CD74, CD82, and CDC42EP5. Genes linked to G(2)/M and germinal center were down-regulated. Comparison of the gene expression profiles of NMZL and FL showed enriched expression of CHIT1, TGFB1, and TACI in NMZL, and BCL6, LMO2, and CD10 in FL. NMZL displayed increased expression of miR-221, miR-223, and let-7f, whereas FL strongly expressed miR-494. Our study identifies new candidate diagnostic molecules for NMZL and reveals survival pathways activated in NMZL.

Splenic follicular lymphoma: clinicopathologic characteristics of a series of 32 cases.

The spleen is frequently involved in B-cell lymphomas other than splenic marginal zone lymphoma. Here we describe a series of follicular lymphoma (FL) cases diagnosed in the spleen, consisting of 32 patients who presented clinically with splenomegaly, and slight or no peripheral lymphadenopathy. The splenic specimen had a micronodular pattern, germinal center cytologic composition, and frequent presence of marginal zone-like cells at the periphery of the nodules. Twenty cases showed absence or only partial/weak bcl2 protein expression, and 12 cases had homogeneous and strong positive bcl2 expression. The incidences of t(14;18)(q32;q21), CD10 expression, low histologic grade, and low proliferative index were significantly more frequent in FL bcl2-positive cases than in FL bcl2-negative cases. Splenic FL cases showed frequent relapses, with an overall survival of 55% at 5 years. No significant differences in survival were found between bcl2-negative and bcl2-positive cases. Splenic FL cases could be divided into 2 main variants: one was similar to classic FL with t(14;18) and CD10 expression, whereas the other was characterized by a higher proliferation index and histologic grade, and was more frequently diagnosed as a disease restricted to the spleen. Recognition of the distinct nature of these tumors should facilitate appropriate studies for determining the best therapy for such cases.

DNA methylation-profiling identifies two splenic marginal zone lymphoma subgroups with different clinical and genetic features

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: comprehensive analysis of gene expression and miRNA profiling.

Splenic marginal zone lymphoma is a small B-cell neoplasm whose molecular pathogenesis is still essentially unknown and whose differentiation from other small B-cell lymphomas is hampered by the lack of specific markers. We have analyzed the gene expression and miRNA profiles of 31 splenic marginal zone lymphoma cases. For comparison, 7 spleens with reactive lymphoid hyperplasia, 10 spleens infiltrated by chronic lymphocytic leukemia, 12 spleens with follicular lymphoma, 6 spleens infiltrated by mantle cell lymphoma and 15 lymph nodes infiltrated by nodal marginal zone lymphoma were included. The results were validated by qRT–PCR in an independent series including 77 paraffin-embedded splenic marginal zone lymphomas. The splenic marginal zone lymphoma miRNA signature had deregulated expression of 51 miRNAs. The most highly overexpressed miRNAs were miR-155, miR-21, miR-34a, miR-193b and miR-100, while the most repressed miRNAs were miR-377, miR-27b, miR-145, miR-376a and miR-424. MiRNAs located in 14q32-31 were underexpressed in splenic marginal zone lymphoma compared with reactive lymphoid tissues and other B-cell lymphomas. Finally, the gene expression data were integrated with the miRNA profile to identify functional relationships between genes and deregulated miRNAs. Our study reveals miRNAs that are deregulated in splenic marginal zone lymphoma and identifies new candidate diagnostic molecules for splenic marginal zone lymphoma.

Circulating tumor DNA reveals genetics, clonal evolution and residual disease in classical Hodgkin lymphoma

The rarity of neoplastic cells in the biopsy imposes major technical hurdles that have so far limited genomic studies in classical Hodgkin lymphoma (cHL). By using a highly sensitive and robust deep next-generation sequencing approach for circulating tumor DNA (ctDNA), we aimed to identify the genetics of cHL in different clinical phases, as well as its modifications on treatment. The analysis was based on specimens collected from 80 newly diagnosed and 32 refractory patients with cHL, including longitudinal samples collected under ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) chemotherapy and longitudinal samples from relapsing patients treated with chemotherapy and immunotherapy. ctDNA mirrored Hodgkin and Reed-Sternberg cell genetics, thus establishing ctDNA as an easily accessible source of tumor DNA for cHL genotyping. By identifying STAT6 as the most frequently mutated gene in ∼40% of cases, we refined the current knowledge of cHL genetics. Longitudinal ctDNA profiling identified treatment-dependent patterns of clonal evolution in patients relapsing after chemotherapy and patients maintained in partial remission under immunotherapy. By measuring ctDNA changes during therapy, we propose ctDNA as a radiation-free tool to track residual disease that may integrate positron emission tomography imaging for the early identification of chemorefractory patients with cHL. Collectively, our results provide the proof of concept that ctDNA may serve as a novel precision medicine biomarker in cHL.

Inhibition of Notch pathway arrests PTEN-deficient advanced prostate cancer by triggering p27-driven cellular senescence.

Activation of NOTCH signalling is associated with advanced prostate cancer and treatment resistance in prostate cancer patients. However, the mechanism that drives NOTCH activation in prostate cancer remains still elusive. Moreover, preclinical evidence of the therapeutic efficacy of NOTCH inhibitors in prostate cancer is lacking. Here, we provide evidence that PTEN loss in prostate tumours upregulates the expression of ADAM17, thereby activating NOTCH signalling. Using prostate conditional inactivation of both Pten and Notch1 along with preclinical trials carried out in Pten-null prostate conditional mouse models, we demonstrate that Pten-deficient prostate tumours are addicted to the NOTCH signalling. Importantly, we find that pharmacological inhibition of γ-secretase promotes growth arrest in both Pten-null and Pten/Trp53-null prostate tumours by triggering cellular senescence. Altogether, our findings describe a novel pro-tumorigenic network that links PTEN loss to ADAM17 and NOTCH signalling, thus providing the rational for the use of γ-secretase inhibitors in advanced prostate cancer patients.

Inhibition of CHK1 and WEE1 as a new therapeutic approach in diffuse large B cell lymphomas with MYC deregulation

Diffuse large B cell lymphoma (DLBCL) is the most common type of aggressive lymphoma. Despite the remarkable progress made with the addition of rituximab to standard chemotherapy (CHOP: doxorubicin, cyclophosphamide, vincristine, prednisone), ~40% of patients present refractory disease or disease that will relapse after initial response. Unfortunately, the majority of patients with relapsed DLBCL will succumb to the disease. Aberrations of the oncogene MYC are common in DLBCL and define patients with a more aggressive phenotype and a poor outcome with an overall survival below 30% after 2 years (Barrans et al, 2010). Moreover, up to 40% of DLBCLs display high MYC protein expression, irrespective of the presence of translocations but still associated with inferior outcome, suggesting that mechanisms other than gene alterations are responsible for high protein expression (Testoni et al, 2015). Thus MYC is a crucial target for DLBCL therapy but the inability to effectively target this transcription factor limits the possibility to find new therapeutic strategies. Synthetic lethal approaches have been expanded to target essential signalling pathways downstream of MYC. The hypothesis that CHK1 protein kinase (also known as CHEK1), by allowing the repair of the insults caused by MYC (endogenous DNA damage from replicative stress) may be a crucial target to inhibit in MYCdriven malignancies has been recently demonstrated (Hoglund et al, 2011; Ravi et al, 2016). CHK1 inhibitors are strongly synergistic with WEE1 inhibitors in many experimental models (Carrassa et al, 2012) and both kinase inhibitors are under early clinical investigation (Daud et al, 2015; Scagliotti et al, 2016). We had recently reported that DLBCL cell lines are more sensitive to the single inhibition of CHK1 and WEE1 inhibitors than epithelial cancer cell lines, albeit at a lesser extent than mantle cell lymphoma (MCL) cell lines (Chila et al, 2015). Here, we investigated the effects of CHK1 and WEE1 inhibition in a panel of DLBCL cell lines (Fig 1A) with different MYC status (Table S1). We did not observe any specific correlation between MYC status and the extent of sensitivity to the CHK1 inhibitor PF-00477736 and the WEE1 inhibitor AZD-1775 (Fig S1). MYC, CHK1 and WEE1 protein levels and the degree of endogenous constitutive DNA damage (cH2AX and pS317CHK1) detected in DLBCL cell lines did not correlate with the sensitivity to PF00477736 or to AZD-1775. Similar results were seen at the mRNA level for MYC, CHEK1 and WEE1. The isobologram (Fig 1B) summarizes the combination index (CI) value of each cell line at the 50% inhibitory concentration IC50 dose when the two drugs were combined and clearly shows a synergistic effect in all the DLBCL cell lines regardless of molecular subtype and of MYC status, suggesting that this drug combination has a strong and broad effect in DLBCLs. A similar synergistic effect was observed by combining the WEE1 inhibitor AZD-1775 with another CHK1 inhibitor (AZD-7762) (data not shown). The strong synergistic effect was associated with cell death by apoptosis as suggested by the detection of increased caspase-3 activity observed after combined treatment (Fig S2). The CI values at IC50 in the different cell lines and their WEE1 protein or mRNA levels negatively correlated (P = 0 0015 and P = 0 0029 respectively) (Fig 1C), while the protein or mRNA levels of CHK1, MYC and constitutive DNA damage did not correlate with the extent of the synergistic effect. A significant correlation with WEE1 was confirmed also when five MCL cell lines were included in the analysis (data not shown). These data suggest that high WEE1 expression levels may be considered a molecular biomarker for the detection of lymphoma patients that could better benefit from this combined treatment. We took advantage of a large series of publicly available gene expression profiles for CHOP and for rituximab plus CHOP treated DLBCL cases (GSE10846) (Lenz et al, 2008) to further investigate the role of CHK1 and WEE1 in DLBCL. WEE1 was expressed at higher levels in germinal centre B (GCB) than activated B cell-like (ABC) DLBCL, while the opposite was true for CHEK1, albeit at lower extent (Fig S3), suggesting that GCB-DLBCL could potentially be more sensitive to the combined therapy with CHK1 and WEE1 inhibitors. The combined treatment induced an increased cH2AX in all the cell lines (Fig 2A). Interestingly, a decreased MYC protein level was observed after combined treatment in all except one cell lines (OCI-Ly10) bearing a wildtype MYC (Fig 2A). In this cell line, despite an observed increased cH2AX, no decreased pCDK2 (Fig 2B) and less activity of the combination in terms of CI (0 77) was observed. A similar decrease in MYC protein levels after this drug combination was observed in MCL cell lines (Fig S4), while CHK1 protein expression remained unchanged (Chila et al, 2015 and data not shown). The striking decrease in MYC protein levels was not associated with a significant decrease in MYC mRNA (Fig 2C). Studies on MYC protein stability in OCI-Ly7 cells treated with the two drugs and then with cycloheximide (CHX), showed that MYC protein Correspondence

Abstract 2651: A novel CD19 targeting antibody-drug conjugate, huB4-DGN462, shows promising in vitro and in vivo activity in CD19-positive lymphoma models

Background. CD19 is a cell surface membrane protein expressed in most mature and immature B cell neoplasms, making it a promising target for antibody-drug conjugate (ADC) therapy for B cell malignancies. Here we describe the preclinical activity of a novel CD19-targeting ADC, based on the potent indolinobenzodiazepine DNA-alkylating payload DGN462. Methods. The humanized anti-CD19 antibody, huB4, was conjugated to DGN462 via a cleavable disulfide linker, sulfo-SPDB. In vitro activity of the huB4-DGN462 ADC or the unconjugated DGN462 toxin was evaluated in 54 lymphoma cell lines [27 diffuse large B cell lymphomas (DLBCL); 10 mantle cell lymphomas; 6 marginal zone lymphomas; 5 anaplastic large T-cell lymphomas; 6 others]. Cell proliferation/viability after 72h of exposure was measured using a MTT assay. Apoptosis activation was defined by at least 1.5-fold increase in caspase 3/7 signal activation in respect to controls using the Promega ApoTox-Glo Triplex Assay. Gene expression profiling (GEP) was performed with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES > |2|, P |1.2|; P Results. huB4-DGN462 was cytotoxic against a broad panel of 48 B cell lymphoma cell lines (median IC50 100 pM; 95%CI, 38-214). The cytotoxic activity was not limited by P53, BCL2, MYC or CDKN2A status, or associated with DLBCL cell of origin. Consistent with overall lower CD19 expression, huB4-DGN462 was significantly (p-value=0.007) less active in eight T cell-derived lymphomas (median IC50 of 1.75 nM (95%CI, 0.5-5.75)) than in B cell lymphomas. huB4-DGN462 induced caspase 3/7 activation in 48/54 cell lines (89%) consistent with an apoptotic mechanism of action. huB4-DGN462 demonstrated compelling anti-tumor activity after a single intravenous dose in two diffuse large B-cell lymphoma cell line xenograft models: DoHH2 (a subcutaneous model) and Farage (a disseminated model). In the DoHH2 model, huB4-DGN462 resulted in a significant, dose-dependent tumor growth delay and survival benefit at 1.7 mg Ab/kg compared to a non-targeted control DGN462 ADC. In the disseminated Farage model, a significant dose-dependent increase in survival was observed in mice treated with as low as 0.17 mg Ab/kg of huB4-DGN462. At 1.7 mg Ab/kg, the life span was increased >400% compared to untreated mice. Conclusions. The novel ADC huB4-DGN462 presented strong preclinical anti-lymphoma activity, which provides evidence for further study. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Alberto J. Arribas, Roberta Pittau Bordone, Andrea Rinaldi, Georg Stussi, Emanuele Zucca, Davide Rossi, Anastasios Stathis, Min Li, Alan Wilhem, Kate Lai, Qifeng Qiu, Stuart Hicks, Callum Sloss, Francesco Bertoni. A novel CD19 targeting antibody-drug conjugate, huB4-DGN462, shows promising in vitro and in vivo activity in CD19-positive lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2651. doi:10.1158/1538-7445.AM2017-2651

PQR309 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in Lymphomas as a Single Agent and in Combination Therapy

Purpose: Activation of the PI3K/mTOR signaling pathway is recurrent in different lymphoma types, and pharmacologic inhibition of the PI3K/mTOR pathway has shown activity in lymphoma patients. Here, we extensively characterized the in vitro and in vivo activity and the mechanism of action of PQR309 (bimiralisib), a novel oral selective dual PI3K/mTOR inhibitor under clinical evaluation, in preclinical lymphoma models. Experimental Design: This study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination, validation experiments on in vivo models and primary cells, proteomics and gene-expression profiling, and comparison with other signaling inhibitors. Results: PQR309 had in vitro antilymphoma activity as single agent and in combination with venetoclax, panobinostat, ibrutinib, lenalidomide, ARV-825, marizomib, and rituximab. Sensitivity to PQR309 was associated with specific baseline gene-expression features, such as high expression of transcripts coding for the BCR pathway. Combining proteomics and RNA profiling, we identified the different contribution of PQR309-induced protein phosphorylation and gene expression changes to the drug mechanism of action. Gene-expression signatures induced by PQR309 and by other signaling inhibitors largely overlapped. PQR309 showed activity in cells with primary or secondary resistance to idelalisib. Conclusions: On the basis of these results, PQR309 appeared as a novel and promising compound that is worth developing in the lymphoma setting. Clin Cancer Res; 24(1); 120–9. ©2017 AACR.

Methylation patterns in marginal zone lymphoma

Promoter DNA methylation is a major regulator of gene expression and transcription. The identification of methylation changes is important for understanding disease pathogenesis, for identifying prognostic markers and can drive novel therapeutic approaches. In this review we summarize the current knowledge regarding DNA methylation in MALT lymphoma, splenic marginal zone lymphoma, nodal marginal zone lymphoma. Despite important differences in the study design for different publications and the existence of a sole large and genome-wide methylation study for splenic marginal zone lymphoma, it is clear that DNA methylation plays an important role in marginal zone lymphomas, in which it contributes to the inactivation of tumor suppressors but also to the expression of genes sustaining tumor cell survival and proliferation. Existing preclinical data provide the rationale to target the methylation machinery in these disorders.