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Supplemental Figure S1. Sequence electropherograms and histopathology from representative cases with CTNNB1, PIK3CA, NRAS, FGFR2, SMARCB1, HRAS and SMO mutations.

Supplementary Data (Fig. S1- S6)

Mature B-cell neoplasms are the fifth most common neoplasm. Due to significant heterogeneity at the clinical and genetic levels, current therapies for these cancers fail to provide long-term cures. The clinical success of proteasome inhibition for the treatment of multiple myeloma and B-cell lymphomas has made the ubiquitin pathway an important emerging therapeutic target. In this study, we assessed the role of the E3 ligase FBXW7 in mature B-cell neoplasms. FBXW7 targeted the frequently inactivated tumor suppressor KMT2D for protein degradation, subsequently regulating gene expression signatures related to oxidative phosphorylation (OxPhos). Loss of FBXW7 inhibited diffuse large B-cell lymphoma cell growth and further sensitized cells to OxPhos inhibition. These data elucidate a novel mechanism of regulation of KMT2D levels by the ubiquitin pathway and uncover a role of FBXW7 in regulating oxidative phosphorylation in B-cell malignancies.Significance:These findings characterize FBXW7 as a prosurvival factor in B-cell lymphoma via degradation of the chromatin modifier KMT2D.

Purpose: Ameloblastoma is an odontogenic neoplasm whose overall mutational landscape has not been well characterized. We sought to characterize pathogenic mutations in ameloblastoma and their clinical and functional significance with an emphasis on the mitogen-activated protein kinase (MAPK) pathway.Experimental Design: A total of 84 ameloblastomas and 40 non-ameloblastoma odontogenic tumors were evaluated with a combination of BRAF V600E allele–specific PCR, VE1 immunohistochemistry, the Ion AmpliSeq Cancer Hotspot Panel, and Sanger sequencing. Efficacy of a BRAF inhibitor was evaluated in an ameloblastoma-derived cell line.Results: Somatic, activating, and mutually exclusive RAS–BRAF and FGFR2 mutations were identified in 88% of cases. Somatic mutations in SMO, CTNNB1, PIK3CA, and SMARCB1 were also identified. BRAF V600E was the most common mutation, found in 62% of ameloblastomas and in ameloblastic fibromas/fibrodentinomas but not in other odontogenic tumors. This mutation was associated with a younger age of onset, whereas BRAF wild-type cases arose more frequently in the maxilla and showed earlier recurrences. One hundred percent concordance was observed between VE1 immunohistochemistry and molecular detection of BRAF V600E mutations. Ameloblastoma cells demonstrated constitutive MAPK pathway activation in vitro. Proliferation and MAPK activation were potently inhibited by the BRAF inhibitor vemurafenib.Conclusions: Our findings suggest that activating FGFR2–RAS–BRAF mutations play a critical role in the pathogenesis of most cases of ameloblastoma. Somatic mutations in SMO, CTNNB1, PIK3CA, and SMARCB1 may function as secondary mutations. BRAF V600E mutations have both diagnostic and prognostic implications. In vitro response of ameloblastoma to a BRAF inhibitor suggests a potential role for targeted therapy. Clin Cancer Res; 20(21); 5517–26. ©2014 AACR.

Rational new strategies are needed to treat tumors resistant to kinase inhibitors. Mechanistic studies of resistance provide fertile ground for development of new approaches. Cancer drug addiction is a paradoxical resistance phenomenon, well-described in MEK-ERK-driven solid tumors, in which drug-target overexpression promotes resistance but a toxic overdose of signaling if the inhibitor is withdrawn. This can permit prolonged control of tumors through intermittent dosing. We and others showed previously that cancer drug addiction arises also in the hematologic malignancy ALK-positive anaplastic large-cell lymphoma (ALCL) resistant to ALK-specific tyrosine kinase inhibitors (TKIs). This is driven by the overexpression of the fusion kinase NPM1-ALK, but the mechanism by which ALK overactivity drives toxicity upon TKI withdrawal remained obscure. Here we reveal the mechanism of ALK-TKI addiction in ALCL. We interrogated the well-described mechanism of MEK/ERK pathway inhibitor addiction in solid tumors and found it does not apply to ALCL. Instead, phosphoproteomics and confirmatory functional studies revealed that the STAT1 overactivation is the key mechanism of ALK-TKI addiction in ALCL. The withdrawal of TKI from addicted tumors in vitro and in vivo leads to overwhelming phospho-STAT1 activation, turning on its tumor-suppressive gene-expression program and turning off STAT3's oncogenic program. Moreover, a novel NPM1-ALK-positive ALCL PDX model showed a significant survival benefit from intermittent compared with continuous TKI dosing. In sum, we reveal for the first time the mechanism of cancer drug addiction in ALK-positive ALCL and the benefit of scheduled intermittent dosing in high-risk patient-derived tumors in vivo.

To implement a vaginal delivery of the second twin simulation program for obstetric and gynecology residents, to reduce maternal-fetal mortality in the management of twin pregnancies in the delivery room.A prospective education program. The session consisted of a theoretical part, a practical part on a mannequin and an evaluation. The model was designed in a simple and reproducible way. The simulation evaluation was done at several levels according to the validated Kirkpatrick model.A tertiary level university maternity hospital.Ten obstetric and gynecology residents participated.The resident's personal feelings about their ability to perform the maneuvers increased significantly after participation in the simulation session. Their technical skills in performing the maneuvers were assessed in a second step. The majority of the participants considered the model realistic and the session useful. All agreed that the simulation was an essential part of their learning process.A simulated vaginal delivery of the second twin session allows residents to be safely trained in these obstetrical maneuvers, which can be difficult to teach and perform.

Mass spectrometry-based techniques now enable the unbiased identification of proteins in complex mixtures including proteins isolated from cells and tissues. These powerful tools permit near-complete annotation of proteins expressed in cells, tissues or organs. Further, these techniques permit the interrogation of the numerous posttranslational modifications that govern cell-specific responses to signaling cues and underlie the functional heterogeneity of cellular composition and contribute to biological complexity. Parallel developments in technologies such as mass cytometry and multicolor ion-beam imaging which permit multi-parameter detection of numerous proteins at the single-cell and in situ level respectively, are poised to radically impact our understanding of the functional and translational importance of proteins in hematologic conditions. Importantly, the field of proteomics is poised to realize the immensely powerful opportunities in integration with genomic information that is being discovered at an unprecedented pace for many hematologic conditions.

A fundamental requirement for growth of rapidly proliferating cells is metabolic adaptation to promote synthesis of biomass1. ATP citrate lyase (ACLY) is a critical enzyme responsible for synthesis of cytosolic acetyl-CoA, the key building component for de novo fatty acid synthesis and links vital pathways such as carbohydrate and lipid metabolism2. The mechanisms of ACLY regulation are not completely understood and the regulation of ACLY function by tyrosine phosphorylation is unknown. Here we show using mass-spectrometry-driven phosphoproteomics and metabolomics that ACLY is phosphorylated and functionally regulated at an evolutionary conserved residue, Y682. Physiologic signals promoting rapid cell growth such as epidermal growth factor stimulation in epithelial cells and T-cell receptor activation in primary human T-cells result in rapid phosphorylation of ACLY at Y682. In vitro kinase assays demonstrate that Y682 is directly phosphorylated by multiple tyrosine kinases, including ALK, ROS1, SRC, JAK2 and LTK. Oncogenically activating structural alterations such as gene-fusions, amplification or point mutations of ALK tyrosine kinase result in constitutive phosphorylation of ACLY in diverse forms of primary human cancer such as lung cancer, anaplastic large cell lymphoma (ALCL) and neuroblastoma. Expression of a phosphorylation-defective ACLY-Y682F mutant in NPM-ALK+ ALCL decreases ACLY activity and attenuates lipid synthesis. Metabolomic analyses reveal that ACLY-Y682F expression results in increased β-oxidation of 13C-oleic acid-labeled fatty acid with increased labeling of +2-citrate (pin vivo. Our results reveal a novel mechanism for direct ACLY regulation that is subverted by multiple oncogenically-activated tyrosine kinases in diverse human cancers. These findings have significant implications for novel therapies targeting ACLY in cancer and metabolism.

We recently demonstrated that obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab compared to rituximab (RTX) mediates significantly enhanced antibody-dependent cell cytotoxicity (ADCC) in vitro and increased overall survival in a Burkitt lymphoma (BL) xenograft non-obese diabetic severe combined immunodeficiency gamma (NSG) model. In this study we compared the phosphoproteomic changes by pathway analysis following obinutuzumab vs RTX against RTX-sensitive (Raji) and -resistant BL (Raji4RH). Phosphoproteomic analyses were performed by mass-spectrometry (MS)-based label-free quantitative phosphoproteomic profiling. We demonstrated that 418 proteins in Raji and 377 proteins in Raji 4RH, were differentially phosphorylated (>1.5-fold) after obinutuzumab vs. RTX. Proteins that were significantly differentially phosphorylated included the B cell antigen receptor (BCR) (PLCG2, BTK and GSK3B), Fc gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF1), and natural killer cell-mediated cytotoxicity (MAPK1, RAF1, PLCG2 and MAPK3) signaling pathways. Differential phosphorylation of BCR or cytotoxicity pathway proteins revealed significant up-regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab compared to RTX. Silencing of PLCG2 in the BCR and MAPK1 in the cytotoxicity pathway significantly increased BL proliferation and decreased BL cytotoxicity after obinutuzumab compared to RTX. These results in combination with our previous results demonstrating a significant improvement in in vitro BL cytotoxicity and in vivo BL survival by obinutuzumab compared to RTX may in part be due to differential effects on selected BL protein signaling pathways.

Introduction: B-cell immunophenotype could be swiftly assessed by flow cytometry on blood samples or bone marrow aspirate specimens. It provides crucial information later refined with histologic, genetic and molecular features to assert accurate diagnosis of chronic B-cell lymphoproliferative disorders (B-CLPD). Besides Matutes score we identified additional useful markers, i.e. CD148 and CD180 to classify mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL), respectively. Furthermore, CD200 is known to be highly expressed in chronic lymphoid leukemia (CLL) while absent in MCL. Hypothesis: The determination of CD148, CD180 and CD200 expression on B-cells by flow cytometry on blood samples and/or bone marrow aspirates could be a potent tool to accurately identify B-CLPD. We postulated the existence of the following specific expression patterns in B-CLPD: CD148 dim/CD180 dim/CD200 bright for CLL, CD148 dim/CD180 dim/CD200 dim for lymphoplasmocytic lymphoma (LPL), CD148 bright/CD180 dim/CD200 neg/dim for MCL and CD148 dim/CD180 bright/CD200 dim for MZL . Methods: In a prospective study we investigated the expression of CD148/CD180/CD200 on B-cells from 673 patients at the time of B-CLPD diagnosis in our hospital from 2014 to 2020. We analyzed 440 blood and 233 bone marrow aspirate specimens using a BD FACSCanto II flow cytometry instrument. Based solely on CD148/CD180/CD200 specific expression patterns we postulated a diagnosis of CLL, LPL, MCL or MZL. These postulated diagnoses were later confronted to the final diagnoses when all histologic, genetic and molecular features were finalized. Sensitivity, specificity, positive and negative predictive values of the expression profiles were determined. In addition, to investigate the relative importance of these three CD markers we then normalized their mean fluorescence intensities (MFI) and applied several supervised machine learning algorithms including Logistic Regression, Random Forest and Light Gradient Boosting Machine (LightGBM). Results: Out of the 673 clinical samples the CD148/CD180/CD200 expression patterns classified 212 specimens as CLL/SLL (30.8%), 160 as LPL (23.8%), 76 as MCL (11.28%) and 169 as MZL (25%). These diagnosis hypotheses were retrospectively compared to the final diagnoses based on all histologic, genetic and molecular features These diagnosis hypotheses of CLL, LPL, MCL and MZL were consistent with the final diagnosis in 583 out of the 617 corresponding cases (94%) with high positive and negative predictive values. The characteristics of the diagnosis accuracy are detailed in the table below. HCL and FL were not further investigated as their immunophenotype usually do not overlap with those of other B-CLPD. Seventeen out of 617 patients (17/617, 5.3%) did not displayed a clear CD148/CD180/CD200 pattern: 9 LPL, 4 CLL and 4 MZL. In sixteen patients (16/617, 5.0%) the diagnosis hypothesis based on this strategy was not confirmed after completion of the exploration including karyotype, MYD88 L265P mutational status, CCND1 overexpression and pathology explorations. We next investigated the relative importance of these 3 markers. We focused on MFI values of CD148, CD180 and CD200 and three categorical "positive or negative" markers (CD5, CD23, FMC7) that were assembled into a composite marker. After Cox-box normalization of CD148, CD180 and CD200 MFIs, a set of supervised machine learning algorithms including Logistic Regression, Random Forest and Light Gradient Boosting Machine (LightGBM) were applied to the cohort of CLL, LPL, MCL and MZL. We established that the highest diagnosis weights were obtained for CD200 in CLL, CD200 and CD148 in MCL (negatively and positively, respectively), CD180 in MZL. In LPL, CD148, CD180 and CD200 had the highest weights using LightGBM and Random Forest algorithms, while Logistic Regression determined that CD5 and CD23 had the highest (negative) weights. In conclusion, the determination of CD148/CD180/CD200 surface expression patterns by flow cytometry, along with morphology, allowed to assert an accurate diagnosis hypothesis in CLL, MCL, LPL and MZL with high positive and negative predictive values. Machine learning algorithms allowed to measure the relative importance of these markers, that could be of great help in case of discordant expression of the main diagnosis markers. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction Initially dispensed in specialized simulation centers, simulation training has recently begun to take place directly in healthcare facilities, that is, in situ. The objective of this study is to assess the effect of training by in situ simulation in obstetrics. Material and methods The training program, dispensed over a 2-day period, took place in maternity units of the members of the Pays de la Loire perinatal network, Reseau Securite Naissance (Network Safety Birth). All participants received a learner satisfaction questionnaire to complete (5-point Likert-like scales). Then, at least 6 months later, each maternity ward received a general questionnaire to assess the effect of the training, as well as a second questionnaire specific to each institution, about the areas for improvement proposed by the teaching team after debriefings. Results The 14 establishments included in our study returned 375 satisfaction questionnaires. In all, 91.1 % were very satisfied and reported that the training met their expectations, and 99.7 % thought the program would have an impact on their professional practice. More than 94 % of the learners wanted more simulation sessions. Among the 14 facilities, 9 (64.3 %) returned their evaluation questionnaires. In 44.4 % of cases, they reported improvement in team cohesion and in team communication, while the others reported these elements remained stable. All maternity units reported that the training had a positive impact on their team, and that they would be interested in new training program with in situ simulation. Discussion Most participants clearly appreciated this training. In situ simulation training also led to the identification of areas for improvements, many of them accomplished, through the drafting of protocols or material modifications aimed at improving staff practices and therefore global patient care. There are many ways by which these training programs can be made sustainable, including the development of a new training program of in situ simulation or the creation of onsite simulation sessions on demand or by the professionals at each institution. Conclusion This survey demonstrated the enthusiasm of healthcare professionals about in situ simulation. Moreover, overall improvement in team communication and cohesion was reported in the medium term (evaluation at more than 6 months). The interest of continuing these training sessions appears undeniable.

Purpose Fusions involving the ALK gene are the predominant genetic lesion underlying pediatric anaplastic large cell lymphomas (ALCL) and inflammatory myofibroblastic tumors (IMTs). We assessed the activity of the ALK inhibitor crizotinib in patients who had no known curative treatment options at diagnosis or with relapsed/recurrent disease. Methods In this study, 26 patients with relapsed/refractory ALK-positive ALCL and 14 patients with metastatic or inoperable ALK-positive IMT received crizotinib orally twice daily. Study objectives were measurement of efficacy and safety. Correlative studies evaluated the serial detection of NPM-ALK fusion transcripts in patients with ALCL. Results The overall response rates for patients with ALCL treated at doses of 165 (ALCL165) and 280 (ALCL280) mg/m2 were 83% and 90%, respectively. The overall response rate for patients with IMT (treated at 100, 165, and 280 mg/m2/dose) was 86%. A complete response was observed in 83% (five of six) of ALCL165, 80% (16 of 20) of ALCL280, and 36% (five of 14) of patients with IMT. Partial response rates were 0% (none of six), 10% (two of 20), and 50% (seven of 14), respectively. The median duration of therapy was 2.79, 0.4, and 1.63 years, respectively, with 12 patients ceasing protocol therapy to proceed to transplantation. The most common drug-related adverse event was decrease in neutrophil count in 33% and 70% of the ALCL165 and ALCL280 groups, respectively, and in 43% of patients with IMT. Levels of NPM-ALK decreased during therapy in most patients with ALCL. Conclusion The robust and sustained clinical responses to crizotinib therapy in patients with relapsed ALCL and metastatic or unresectable IMT highlight the importance of the ALK pathway in these diseases.

Identification of biomarkers and therapeutic targets is a critical goal of precision medicine. N-glycoproteins are a particularly attractive class of proteins that constitute potential cancer biomarkers and therapeutic targets for small molecules, antibodies, and cellular therapies. Using mass spectrometry (MS), we generated a compendium of 1,091 N-glycoproteins (from 40 human primary lymphomas and cell lines). Hierarchical clustering revealed distinct subtype signatures that included several subtype-specific biomarkers. Orthogonal immunological studies in 671 primary lymphoma tissue biopsies and 32 lymphoma-derived cell lines corroborated MS data. In anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL), integration of N-glycoproteomics and transcriptome sequencing revealed an ALK-regulated cytokine/receptor signaling network, including vulnerabilities corroborated by a genome-wide clustered regularly interspaced short palindromic screen. Functional targeting of IL-31 receptor β, an ALCL-enriched and ALK-regulated N-glycoprotein in this network, abrogated ALK+ALCL growth in vitro and in vivo. Our results highlight the utility of functional proteogenomic approaches for discovery of cancer biomarkers and therapeutic targets.

Mature B-cell neoplasms are the fifth most common neoplasm. Due to significant heterogeneity at the clinical and genetic levels, current therapies for these cancers fail to provide long-term cures. The clinical success of proteasome inhibition for the treatment of multiple myeloma and B-cell lymphomas has made the ubiquitin pathway an important emerging therapeutic target. In this study, we assessed the role of the E3 ligase FBXW7 in mature B-cell neoplasms. FBXW7 targeted the frequently inactivated tumor suppressor KMT2D for protein degradation, subsequently regulating gene expression signatures related to oxidative phosphorylation (OxPhos). Loss of FBXW7 inhibited diffuse large B-cell lymphoma cell growth and further sensitized cells to OxPhos inhibition. These data elucidate a novel mechanism of regulation of KMT2D levels by the ubiquitin pathway and uncover a role of FBXW7 in regulating oxidative phosphorylation in B-cell malignancies. Significance: These findings characterize FBXW7 as a prosurvival factor in B-cell lymphoma via degradation of the chromatin modifier KMT2D.

Purpose: Peripheral T-cell lymphomas are clinically aggressive and usually fatal, as few complete or durable remissions are achieved with currently available therapies. Recent evidence supports a critical role for lymphoma-associated macrophages during T-cell lymphoma progression, but the specific signals involved in the cross-talk between malignant T cells and their microenvironment are poorly understood. Colony-stimulator factor 1 receptor (CSF1R, CD115) is required for the homeostatic survival of tissue-resident macrophages. Interestingly, its aberrant expression has been reported in a subset of tumors. In this article, we evaluated its expression and oncogenic role in T-cell lymphomas. Experimental Design: Loss-of-function studies, including pharmacologic inhibition with a clinically available tyrosine kinase inhibitor, pexidartinib, were performed in multiple in vitro and in vivo models. In addition, proteomic and genomic screenings were performed to discover signaling pathways that are activated downstream of CSF1R signaling. Results: We observed that CSF1R is aberrantly expressed in many T-cell lymphomas, including a significant number of peripheral and cutaneous T-cell lymphomas. Colony-stimulating factor 1 (CSF1), in an autocrine or paracrine-dependent manner, leads to CSF1R autophosphorylation and activation in malignant T cells. Furthermore, CSF1R signaling was associated with significant changes in gene expression and in the phosphoproteome, implicating PI3K/AKT/mTOR in CSF1R-mediated T-cell lymphoma growth. We also demonstrated that inhibition of CSF1R in vivo and in vitro models is associated with decreased T-cell lymphoma growth. Conclusions: Collectively, these findings implicate CSF1R in T-cell lymphomagenesis and have significant therapeutic implications.

The spectrum of cutaneous CD30-positive lymphoproliferative disorders (LPDs) includes lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma. Chromosomal translocations targeting tyrosine kinases in CD30-positive LPDs have not been described. Using whole-transcriptome sequencing, we identified a chimeric fusion involving NPM1 (5q35) and TYK2 (19p13) that encodes an NPM1-TYK2 protein containing the oligomerization domain of NPM1 and an intact catalytic domain in TYK2. Fluorescence in situ hybridization revealed NPM1-TYK2 fusions in 2 of 47 (4%) primary cases of CD30-positive LPDs and was absent in other mature T-cell neoplasms (n = 151). Functionally, NPM1-TYK2 induced constitutive TYK2, signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5 activation. Conversely, a kinase-defective NPM1-TYK2 mutant abrogated STAT1/3/5 signaling. Finally, short hairpin RNA-mediated silencing of TYK2 abrogated lymphoma cell growth. This is the first report of recurrent translocations involving TYK2, and it highlights the novel therapeutic opportunities in the treatment of CD30-positive LPDs with TYK2 translocations.

Purpose: Ameloblastoma is an odontogenic neoplasm whose overall mutational landscape has not been well characterized. We sought to characterize pathogenic mutations in ameloblastoma and their clinical and functional significance with an emphasis on the mitogen-activated protein kinase (MAPK) pathway. Experimental Design: A total of 84 ameloblastomas and 40 non-ameloblastoma odontogenic tumors were evaluated with a combination of BRAF V600E allele–specific PCR, VE1 immunohistochemistry, the Ion AmpliSeq Cancer Hotspot Panel, and Sanger sequencing. Efficacy of a BRAF inhibitor was evaluated in an ameloblastoma-derived cell line. Results: Somatic, activating, and mutually exclusive RAS–BRAF and FGFR2 mutations were identified in 88% of cases. Somatic mutations in SMO, CTNNB1, PIK3CA, and SMARCB1 were also identified. BRAF V600E was the most common mutation, found in 62% of ameloblastomas and in ameloblastic fibromas/fibrodentinomas but not in other odontogenic tumors. This mutation was associated with a younger age of onset, whereas BRAF wild-type cases arose more frequently in the maxilla and showed earlier recurrences. One hundred percent concordance was observed between VE1 immunohistochemistry and molecular detection of BRAF V600E mutations. Ameloblastoma cells demonstrated constitutive MAPK pathway activation in vitro. Proliferation and MAPK activation were potently inhibited by the BRAF inhibitor vemurafenib. Conclusions: Our findings suggest that activating FGFR2–RAS–BRAF mutations play a critical role in the pathogenesis of most cases of ameloblastoma. Somatic mutations in SMO, CTNNB1, PIK3CA, and SMARCB1 may function as secondary mutations. BRAF V600E mutations have both diagnostic and prognostic implications. In vitro response of ameloblastoma to a BRAF inhibitor suggests a potential role for targeted therapy. Clin Cancer Res; 20(21); 5517–26. ©2014 AACR.

The comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5B in 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RG mutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5B have not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5B mutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia.

Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.

The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely understood. Here we show using an integrated phosphoproteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphorylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis.

Mechanisms by which NPM-ALK signaling regulates cell migration, invasion and contributes to the oncogenesis of anaplastic large cell lymphoma (ALCL) are not completely understood. In an attempt to identify novel actin signaling pathways regulated by NPM-ALK, a comprehensive phosphoproteome analysis of ALCL cell lines was performed in the presence or absence of NPM-ALK activity. Numerous phosphoproteins involved in actin dynamics including Wiskott-Aldrich syndrome protein (WASp) were regulated by NPM-ALK. Network analysis revealed that WASp is a central component of the NPM-ALK-dependent actin signaling pathway. Here we show that NPM-ALK phosphorylates WASp at its known activation site (Y290) as well as at a novel residue (Y102). Phosphorylation of WASp at Y102 negatively regulates its interaction with Wiskott-Aldrich interacting protein and decreases its protein stability. Phosphorylation of WASp at Y102 enhances anchorage-independent growth and tumor growth in an in vivo xenograft model and enhances invasive properties of ALCL. We show that knock-down of WASp or expression of Y102F mutant of WASp decreases colony formation and in vivo tumor growth. Our results show that WASp is a novel substrate of ALK and has a critical role in regulating invasiveness and oncogenesis of ALCL.

This chapter provides an overview of potential and current clinical applications of proteomics. Although the clinical applications of protein microarrays are discussed in this chapter, we particularly emphasize the potential utilization of mass spectrometry-based approaches for diagnosis, biomarker detection, and quantitation.

Le lymphome a cellule du manteau est une proliferation lymphoide B maligne tres heterogene mais tres bien definie par la presence d’une translocation caracteristique, la translocation t(11 ;14)(q13 ;q32) deregulant la cycline D1, proteine majeure du cycle cellulaire de la phase de transition G1-S. Ce lymphome est caracterise par une evolution clinique rapidement pejorative et une chimioresistance aux traitements classiques. Les mecanismes moleculaires et genetiques impliques dans sa physiopathologie associent une deregulation de la proliferation cellulaire et des signaux de survie cellulaire, ainsi qu’une deregulation des genes de chimioresistance, avec un haut degre d’instabilite genetique semblant lie a un defaut de la reponse aux alterations de l’ADN. La comprehension de ces mecanismes et de leur consequence sur la cellule du manteau est la base du rationnel des nouvelles strategies therapeutiques qui pourront etre proposees aux patients presentant un lymphome a cellules du manteau.

Sézary syndrome (SS) is an aggressive leukaemia of mature T cells with poor prognosis and limited options for targeted therapies. The comprehensive genetic alterations underlying the pathogenesis of SS are unknown. Here we integrate whole-genome sequencing (n=6), whole-exome sequencing (n=66) and array comparative genomic hybridization-based copy-number analysis (n=80) of primary SS samples. We identify previously unknown recurrent loss-of-function aberrations targeting members of the chromatin remodelling/histone modification and trithorax families, including ARID1A in which functional loss from nonsense and frameshift mutations and/or targeted deletions is observed in 40.3% of SS genomes. We also identify recurrent gain-of-function mutations targeting PLCG1 (9%) and JAK1, JAK3, STAT3 and STAT5B (JAK/STAT total ∼11%). Functional studies reveal sensitivity of JAK1-mutated primary SS cells to JAK inhibitor treatment. These results highlight the complex genomic landscape of SS and a role for inhibition of JAK/STAT pathways for the treatment of SS., Sézary syndrome is a T cell malignancy that has been poorly characterized at the genome level. In this study, Kiel et al. perform whole-genome analyses and identify mutations in the JAK–STAT pathway and show that primary cells are sensitive to JAK inhibitors.

Inverted sinonasal papilloma (ISP) is a locally aggressive neoplasm associated with sinonasal squamous cell carcinoma (SNSCC) in 10% to 25% of cases. To date, no recurrent mutations have been identified in ISP or SNSCC. Using targeted next-generation sequencing and Sanger sequencing, we identified activating EGFR mutations in 88% of ISP and 77% of ISP-associated SNSCC. Identical EGFR genotypes were found in matched pairs of ISP and associated SNSCC, providing the first genetic evidence of a biologic link between these tumors. EGFR mutations were not identified in exophytic or oncocytic papillomas or non–ISP-associated SNSCC, suggesting that the ISP/SNSCC spectrum is biologically distinct among sinonasal squamous tumors. Patients with ISP harboring EGFR mutations also exhibited an increased progression-free survival compared with those with wild-type EGFR. Finally, treatment of ISP-associated carcinoma cells with irreversible EGFR inhibitors resulted in inactivation of EGFR signaling and growth inhibition. These findings implicate a prominent role for activating EGFR mutations in the pathogenesis of ISP and associated SNSCC and rationalize consideration of irreversible EGFR inhibitors in the therapy of these tumors. Cancer Res; 75(13); 2600–6. ©2015 AACR.

To determine the steady-state trough serum and epithelial lining fluid (ELF) concentrations of teicoplanin 12 mg/kg per day in critically ill patients with ventilator associated pneumonia.Prospective, pharmacokinetic study in the surgical intensive care unit in a university hospital.Thirteen adult patients with nosocomial bacterial pneumonia on mechanical ventilation were enrolled.All subjects received a 30-min intravenous infusion of 12 mg/kg teicoplanin every 12 h for 2 consecutive days followed by 12 mg/kg once daily. Teicoplanin concentrations in serum and ELF were determined simultaneously 4-6 days after antibiotic administration started.The median total and free concentrations of teicoplanin in serum at trough were 15.9 microg/ml (range 8.8-29.9) and 3.7 (2.0-5.4), respectively. The concentration in ELF was 4.9 (2.0-11.8).In critically ill patients with ventilator-associated pneumonia the administration of high teicoplanin doses is required to reach sufficient trough antibiotic concentrations in lung tissues at steady state. At that time trough-free concentrations of teicoplanin in serum and ELF are comparable.

Opioids may activate pain facilitatory systems opposing analgesia. We investigated whether large-dose remifentanil given during IV anesthesia caused postoperative morphine overconsumption and whether nefopam (a centrally acting analgesic) could reduce this. Sixty patients scheduled for abdominal surgery were included in this prospective, randomized study. The first 30 patients received either small-dose (Group S: 3 ng/mL) or large-dose (Group L: 8 ng/mL) remifentanil administrated by a target-controlled infusion during propofol anesthesia. Before skin closure, patients received morphine 0.15 mg/kg. Another 30 patients also received nefopam 20 mg intraoperatively. Postoperative pain was controlled by titration of morphine, followed by patient-controlled morphine analgesia (PCA). Morphine was requested earlier in Group L than in Group S (10 [1-63] min versus 37 [5-90] min, median [range]; P < 0.002). The dose of morphine by titration was larger in Group L than in Group S (0.28 [0.04-0.38] mg/kg versus 0.16 [0.03-0.41] mg/kg; P < 0.05). PCA morphine consumption and pain scores were similar. There were no differences between the nefopam groups in the time to first morphine request or in the dose of morphine by titration. Postoperative morphine overconsumption occurred after large-dose remifentanil and propofol anesthesia during the early postoperative period. Pretreatment with nefopam could be useful to prevent pain sensitization induced by opioids.

Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of hematological malignancies; however, the extent to which deregulated phosphorylation may be involved in B-cell non-Hodgkin lymphoma (B-NHL) pathogenesis is largely unknown. To identify phosphorylation events important in B-NHLs, we performed mass spectrometry–based, label-free, semiquantitative phosphoproteomic profiling of 11 cell lines derived from three B-NHL categories: Burkitt lymphoma, follicular lymphoma, and mantle-cell lymphoma. In all, 6579 unique phosphopeptides, corresponding to 1701 unique phosphorylated proteins, were identified and quantified. The data are available via ProteomeXchange with identifier PXD000658. Hierarchical clustering highlighted distinct phosphoproteomic signatures associated with each lymphoma subtype. Interestingly, germinal center–derived B-NHL cell lines were characterized by phosphorylation of proteins involved in the B-cell receptor signaling. Of these proteins, phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) was identified with the most phosphorylated tyrosine peptides in Burkitt lymphoma and follicular lymphoma. PAG1 knockdown resulted in perturbation of the tyrosine phosphosignature of B-cell receptor signaling components. Significantly, PAG1 knockdown increased cell proliferation and response to antigen stimulation of these germinal center–derived B-NHLs. These data provide a detailed annotation of phosphorylated proteins in human lymphoid cancer. Overall, our study revealed the utility of unbiased phosphoproteome interrogation in characterizing signaling networks that may provide insights into pathogenesis mechanisms in B-cell lymphomas.

This chapter provides an introduction to proteomics and describes current applications of proteomic technologies in investigative and clinical settings. Mass spectrometry-based proteomics is emphasized as a technology for unbiased and targeted interrogation of complex protein mixtures. Technological advances now permit higher sensitivity and accuracy for detection and quantification of biomarkers. Perspectives on current and future applications of mass spectrometry-based proteomics are provided in the context of diagnostic laboratory testing.

It has been shown that non-aqueous capillary electrophoresis (NACE) can provide improved separations in comparison to those obtained using conventional CE under aqueous conditions [1] (ACE). Previous work carried out in our laboratories involving initial investigations into the technique have been reported [2]. Based on the findings of that work it was possible to separate a variety of basic pharmaceuticals from selected impurities and to obtain the successful separation of some hydrophobic sulphonic acids. The successful coupling of NACE to mass spectrometry (NACE-MS) has also been demonstrated.

Background: Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007) and further improved with the addition of rituximab (Goldman/Cairo et al, Leukemia, 2013, Cairo et al. JCO, 2012). However, a subset of patients with chemoimmunotherapy resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012, Barth et al. BJH, 2013). Obinutuzumab, a novel glycoengineered type II CD20 Ab, mediates enhanced cell death & ADCC against B-cell lymphoma vs. RTX (Awasthi/Cairo et al. BJH, 2015), and was recently FDA and EMA approved for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathway altered differentially following obinutuzumab vs RTX against RTX-sensitive/resistant BL Methods: Raji (CD20+) and Raji-4RH) cells were cultured in RPMI with 10% FBS. Tumor cells were incubated with 100 μg/ml obinutuzumab, and/or RTX for 24 hrs. For phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji /Raji4RH in the presence/absence of obinutuzumab or rituximab or isotype control. Silencing of PLCG2 (Dharmacon, USA) and MAPK1 (Sigma Aldrich, USA) in Raji/Raji4RH cell lines was carried out according to the manufacturer's instructions Results: In all 978 unique phosphorylated proteins were identified. Out of these 661 phosphoproteins were identified after obinutuzumab vs. 615 in RTX treatment, respectively. For the Raji4RH, 534 phosphoproteins were identified after obinutuzumab and 534 in RTX treatment, respectively (Fig.1). Functional annotation of proteins differentially phosphorylated in response to obinutuzumab vs. RTX (>1.5-fold) reveals the involvement of the BCR (PLCG2, BTK & GSK3B), FC gamma phagocytosis (FCRG2B, MAPK1 & RAF1), and Natural killer cell-mediated cytotoxicity (MAPK1, RAF1& PLCG2) signaling pathways (Fig. 2). Differential phosphorylations of proteins involved in BCR or cytotoxicity pathways were validated by western blot after incubation with obinutuzumab vs. RTX in Raji/ Raji4RH cell lines, revealed up regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab vs. RTX treatment in Raji. Silencing, of PLCG2 and MAPK1 pathway, significantly increased cell proliferation and decreased cytotoxicity after obinutuzumab treatment in Raji (P = 0.0002 & 0.000002) but no change in Raji4RH. Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, and cytotoxicity signaling pathways. Obinutuzumab function differentially in RTX resistant and sensitive BL cell lines which may provide insights into alternate therapeutic strategy in RTX resistant BL. Citation Format: Aradhana Awasthi Tiwari, Delphine C.m. Rolland, Mona Elmacken, Janet Ayello, Lisa Kurien, Carmella van de Ven, Venkatesha Basrur, Kevin Conlon, Damine Fermin, Matthew J. Barth, Christian Klein, Kojo S.j. Elenitoba-Johnson, Megan Lim, Mitchell S. Cairo. Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3893.

Background: Protein glycosylation is one of the most common posttranslational modifications and plays important roles in many biological processes. N-glycosylation facilitates protein trafficking to membranes and their secretion into the extracellular environment. N-glycoproteins are already used as routine biomarkers in clinical practice. These include cluster of differentiation (CD) proteins that define hematopoietic sub-populations and hematologic neoplasms. These proteins also represent important targets for therapeutic antibodies and immunotoxins. Although N-glycoproteins represent an appealing reservoir of potential biomarkers and therapeutic targets, their expression signatures in lymphoid neoplasia has not been established. Hypothesis: Unbiased characterization of N-glycoproteins of lymphoma cells from different subtypes of lymphoid neoplasia would constitute a valuable resource for identifying rational candidate biomarkers and therapeutic targets. Materials and Methods: We performed solid phase extraction of glycoproteins followed by identification of deglycosylated peptides using LC-MS/MS on 36 well-characterized cell lines representing 14 different human lymphoid neoplasm subtypes. Label-free spectral counting was used to quantify the relative abundance of N-glycoproteins. In addition, the N-glycoproteome profiles of 8 patient-derived lymphoma samples were compared in a blinded fashion to the profiles of the 36 cell lines. We employed orthogonal methods such as western blotting, flow cytometry and immunohistochemistry on cell lines and primary human lymphoma biopsies to validate several candidates. Pharmacological inhibition and RNAi-mediated silencing were used to functionally validate glycoproteins specific to ALK-positive anaplastic large cell lymphoma (ALCL, ALK+). Tumorigenicity was assessed in xenografts in SCID-BEIGE mice. Results: In all, 1,155 unique N-glycoproteins were identified from the analysis of the 36 cell lines. Of note, we identified 188 out of the 363 CD proteins listed by the Human Cell Differentiation Molecules consortium. Importantly, we detected virtually all CD proteins currently used for diagnostic evaluation of lymphoid neoplasia. Unsupervised hierarchical clustering of N-glycoproteomic data accurately designated the 36 cell lines according to their respective lineages, cells of origin and appropriate World Health Organization subtypes. Pearson's correlation between the 36 cell lines and the 8 primary biopsies demonstrated that N-glycoproteomic profiles accurately segregated all primary lymphoma samples within their original diagnostic categories. Using tissue microarrays of 644 primary biopsies of mantle cell lymphoma (n=34), follicular lymphoma (n=183), diffuse large B-cell lymphoma (n=245), classical Hodgkin lymphoma (n=150) and ALCL, ALK+ (n=32) we validated the differential expression of CD44, CD276 and paraoxonase 2 (PON2) in mantle cell lymphoma. In addition, our analyses revealed a distinctive cytokine network signature associated with ALCL, ALK+. The evaluation of CD25 expression in a well-characterized cohort of primary lymphoma biopsies (n=28) consisting of ALCL, ALK+ and ALCL, ALK- demonstrated statistically significant correlation between ALK and CD25 expression (p=0.002). In vitro, functional targeting of CD25 using a toxin-conjugated ligand abrogated ALCL, ALK+ cell growth and promoted cell death by apoptosis. This cytokine network signature also included the selective expression of oncostatin M receptor (OSMR) in ALCL, ALK+. We validated OSMR selective expression in ALCL, ALK+ cells by western blot and flow cytometry. Immunohistochemistry on 56 ALCL biopsies (ALK+ and ALK-) revealed a significant correlation between ALK and OSMR expression (p Conclusion: Our data reveal that several glycoproteins demonstrate characteristic and unique expression signatures in distinct forms of human malignant lymphoid neoplasia. Our results indicate that comprehensive profiling of N-glycoproteomes of cells offers opportunities for discovery of novel lymphoma biomarkers and therapeutic targets. Disclosures No relevant conflicts of interest to declare.

Background: Burkitt Lymphoma (BL) is the most common form of NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007, Cairo et al. JCO, 2012). The prognosis has improved with the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013, Goldman/Cairo et al. BJH, 2014). However, a subset of patients with chemoimmunotherapy-resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however, the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab vs. RTX, a Type I CD20 Ab, mediates enhanced cell death & ADCC against diffuse B-cell lymphoma vs. RTX (Mössner et al. Blood, 2010), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathways are differentially altered following obinutuzumab vs RTX against RTX-sensitive/resistant BL. Methods: Raji (CD20+, ATCC, Manhass, VA) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs. For Phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji/Raji4RH in the presence/absence of obinutuzumab or rituximab (100µg/ml for 24h) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides and subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation. An LTQ Orbitrap XL, in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data that were searched with the Swissprot Human taxonomic protein database (McDonnell and Lim et al, Blood, 2013). Silencing of PLCG2 in Raji and Raji4RH cell lines was carried out according to the manufacturer's instructions (Dharmacon, PA, USA). Results: Four hundred and eighteen out of total 661 proteins in Raji and 377 out of total 534 proteins in Raji4RH were differentially phosphorylated (>1.5 fold) after obinutuzumab treatment. Of these proteins, 46 were expressed at significantly higher levels in obinutuzumab vs. RTX in Raji (Figure 1). However, Raji4RH, the RTX resistant cell line did not show significant increase in phosphorylation of protein following in obinutuzumab vs. RTX. Proteins differentially phosphorylated in response to obinutuzumab vs. RTX are involved in the BCR (PLCG2, BTK, GSK3B and RAF-1), FC gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF-1), and Natural Killer cell-mediated cytotoxicity (MAPK1, RAF-1, PLCG2 and MAPK3) signaling pathways in Raji vs. Raji4RH (Figure 2). Differential phosphorylation of BCR signaling pathways proteins (BTK, PLCG2 and GSK3B), validated by western blot studies after incubation with obinutuzumab vs. RTX in Raji/Raji4RH cell lines, revealed up-regulation of BTK and PLCY2 after obinutuzumab treatment vs. RTX treatment in Raji BL cell line. Silencing one of the BCR signaling pathway protein, PLCG2 significantly increased cell proliferation and decreased cell death after obinutuzumab vs. RTX treatment in Raji (P=0.0001 & 0.004) however, there was no change in Raji 4RH RTX resistant cell line. Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, phagocytosis and cytotoxicity signaling pathways in BL. Knockdown of PLCG2 significantly enhanced BL proliferation and reduced cell death after obinutuzumab vs. RTX treatment. These results offer insights into alternate therapeutic strategies in the treatment of RTX resistant BL. Disclosures Klein: Roche: Employment.

Introduction: Anaplastic large cell lymphoma (ALCL) is characterized by recurrent translocations involving anaplastic lymphoma kinase (ALK) and resulting in oncogenic chimeric fusion tyrosine kinases such as NPM-ALK. We integrated mass-spectrometry-driven phosphoproteomics and metabolomics of ALK+ ALCL and discovered that ATP citrate lyase (ACLY) is a candidate substrate of NPM-ALK at residue Y682. ACLY is a critical enzyme responsible for synthesis of acetyl-CoA and connects vital biosynthetic pathways such as carbohydrate and lipid metabolism. Functional regulation of ACLY by tyrosine phosphorylation is unknown and the significance of tyrosine phosphorylation of ACLY on cancer cell growth and metabolism have never been described. Methods: To identify novel substrates of NPM-ALK, we performed phospho-proteomic analyses of 3 ALK+ALCL derived cell lines. The MS-phosphoproteomic data were subjected to custom bioinformatic algorithms to identify highly statistically correlated phosphotyrosine proteins and phosphosites regulated by ALK kinase activity. We generated a phosphospecific antibody against a highly-ranked candidate (pACLY Y682) and utilized it in western blot analyses on ALK+ and ALK- ALCL lymphoma cell lines. Immunohistochemistry (IHC) on well-characterized primary human ALK+ (n=20) and ALK-ALCL biopsies (n=28) were performed. Co-immunoprecipitations (Co-IP) with HA-tagged ACLY-WT and ACLY-Y682F and ALK antibodies were performed and analyzed by western blot using the pACLY Y682 antibody. In vitro kinase assays were performed to investigate the direct role of NPM-ALK in phosphorylation of ACLY at Y682 using GST-tagged ACLY-WT or ACLY-Y682F peptides in the presence or absence of immunocomplexes containing active NPM-ALK or the kinase-defective K210R mutant. To study the impact of Y682F mutant on lipid metabolism, we generated ALCL-derived cell lines stably transduced to express GFP-tagged ACLY-WT or ACLY-Y682F and subjected them to metabolomic analysis for flux, β-oxidation or lipid synthesis using liquid chromatography/tandem mass spectrometry or gas chromatography platforms. The Seahorse XF24 Flux analyzer was used to measure oxygen consumption rates (OCR) in DEL stably expressing ACLY-WT and ACLY-Y682F. Cells stably expressing ACLY-WT and ACLY-Y682F were evaluated for cell proliferation, colony formation and tumor formation in SCID-BEIGE mouse xenograft models. Results: Phosphoproteomic analyses yielded a dataset containing a total of 626 tyrosine phosphoproteins. Bioinformatic analysis identified pACLY Y682 as a novel candidate substrate of NPM-ALK. Western blot analysis corroborated phosphoproteomic studies and demonstrated that phosphorylation of ALCY at Y682 is regulated by NPM-ALK activity. Expression of active NPM-ALK increased the phosphorylation of ACLY at Y682 in HEK293T cells compared to the K210R mutant. In vitro kinase assays demonstrated that NPM-ALK phosphorylates ACLY at Y682. Immunoprecipitation (IP) of ALK and co-IP (HA) demonstrated interaction of NPM-ALK and ACLY in co-transfected HEK293T cell lysates. Stable expression of ACLY-Y682F or pharmacologic antagonism of ALK with crizotinib resulted in decreased ACLY activity, decreased lipid synthesis and increased fatty acid β-oxidation when compared to ACLY-WT. β-oxidation of 13 C-oleic acid-labeled fatty acid demonstrated increased labeling of +2-citrate (p Conclusion: We identify for the first time a direct tyrosine kinase-mediated mechanism for ACLY regulation. In this regard, our studies demonstrate a novel oncogenic mechanism whereby NPM-ALK controls lipid synthesis and b-oxidation by phosphorylation of ACLY at Y682 and this mechanism contributes to ALCL pathogenesis. Our findings have significant implications for novel therapies targeting tumor metabolism in ALCLs. Disclosures No relevant conflicts of interest to declare.

Mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), and marginal zone lymphoma (MZL) are small B-cell non-Hodgkin lymphomas (NHLs) that may be difficult to distinguish. In order to identify specific proteomic biomarkers, differential proteomic analysis of these three NHLs was performed using surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). Whole cell lysates obtained from 18 MCL, 20 SLL, and 20 MZL biopsies were applied on two different ProteinChips (cationic and anionic). Hierarchical clustering and discriminating scores combined with an innovative bio-informatics microdissection strategy allowed us to distinguish specific lymphoma proteomic signatures based on the expression of 37 protein peaks. SELDI-assisted protein purification combined with nano-liquid chromatography (LC) quadrupole-time of flight tandem mass spectrometry (Q-TOF MS/MS) was used to identify proteins overexpressed in both MCL and SLL tumors. Among them two histones, H2B and H4, were identified in MCL tumor biopsies and the signal recognition particle 9 kDa protein, SRP9, in SLL tumor biopsies.

International audience; Gliomas such as oligodendrogliomas (ODG) and glioblastomas (GBM) are brain tumours with different clinical outcomes. Histology-based classification of these tumour types is often difficult. Therefore the first aim of this study was to gain microRNA data that can be used as reliable signatures of oligodendrogliomas and glioblastomas. We investigated the levels of 282 microRNAs using membrane-array hybridisation and real-time PCR in ODG, GBM and control brain tissues. In comparison to these control tissues, 26 deregulated microRNAs were identified in tumours and the tissue levels of seven microRNAs (miR-21, miR-128, miR-132, miR-134, miR-155, miR-210 and miR-409-5p) appropriately discriminated oligodendrogliomas from glioblastomas. Genomic, epigenomic and host gene expression studies were conducted to investigate the mechanisms involved in these deregulations. Another aim of this study was to better understand glioma physiopathology looking for targets of deregulated microRNAs. We discovered that some targets of these microRNAs such as STAT3, PTBP1 or SIRT1 are differentially expressed in gliomas consistent with deregulation of microRNA expression. Moreover, MDH1, the target of several deregulated microRNAs, is repressed in glioblastomas, making an intramitochondrial-NAD reduction mediated by the mitochondrial aspartate-malate shuttle unlikely. Understanding the connections between microRNAs and bioenergetic pathways in gliomas may lead to identification of novel therapeutic targets.

International audience; INTRODUCTION: The cytotoxic activity of the farnesyltranseferase inhibitor R115777 was evaluated in cell lines representative of mantle cell lymphoma (MCL). METHODS: Cell growth, proliferation, and apoptosis were analyzed in four human MCL cell lines (Granta, NCEB, REC, and UPN1) in presence of R115777, alone or in combination with vincristin, doxorubicin, bortezomib, cisplatin and cytarabine. Inhibition of farnesylation was determined by the appearance of prelamin A. The antitumor activity of R115777, administered p.o. at 100, 250 and 500 mg/kg, was determined in vivo in nude mice xenografted with UPN1 cells. RESULTS: R115777 inhibited the growth of MCL cell lines in vitro with inhibitory concentrations ranging between 2 and 15 nM. A fifty percent decrease of cell viability was observed at concentrations comprised between 0.08 and 17 microM. Apoptosis, evaluated by annexin V and activated caspase 3 staining, was induced in all cell lines, in 40 to 71% of the cells depending on the cell lines. In addition, R115777 significantly increased the cytotoxic effect of vincristine, doxorubicin, bortezomib, cisplatin and cytarabine (p=0.001, p=0.016, p=0.006, p=0.014 and p=0.007 respectively). Exposure of MCL cell lines to R115777 during 72 hours resulted in inhibition of protein farnesylation. R115777 administered p.o. twice daily for 8 consecutive days to mice bearing established s.c. UPN1 xenograft displayed cytostatic activity at the 500 mg/kg dosage. CONCLUSION: We have demonstrated that inhibition of farnesyltransferase by R115777 was associated with growth inhibition and apoptosis of MCL cell lines in vitro and tumor xenograft stability in vivo.

The presence of circulating villous lymphocytes (VLs) in lymphoma patients usually points to splenic marginal zone B-cell lymphoma (SMZL), even if the VLs can be found occasionally in other small B-cell lymphomas. However, those cells are variably described, and detailed cytologic characterization is often lacking. We identified lymphoma cases with numerous basophilic VLs among the large group of splenic lymphoma with VLs, and for further delineation, 37 cases with this particular cytology were analyzed. Patients, predominantly older men, presented with moderate lymphocytosis and splenomegaly without pancytopenia. The monoclonal B cells expressed IgM + D, IgM + G, IgM or IgG, as well as CD76 and CD11c, frequently CD103, and rarely CD123. Spleen sections were peculiar, with atrophic white pulp and a monomorphic diffuse lymphoma infiltration in a congested red pulp. Bone marrow infiltration was interstitial and intrasinusoidal without extensive fibrosis. Cytogenetic analysis showed a frequent absence of clonal aberrations (68%). Most cases (79%) were IgH mutated, with an overrepresentation of VH3 and VH4 gene families. These results, as well as the clinical evolution, show that those lymphoma cases represent a homogeneous group distinct from SMZL and reminiscent of hairy cell leukemia variant, perhaps corresponding to a separate lymphoma entity.

Multi-agent chemotherapy regimens have significantly improved long-term survival of pediatric Burkitt Lymphoma (BL) to over 80%. However, patients with refractory or relapsed disease have a dismal prognosis, stressing the need to identify mechanisms of therapeutic resistance and develop novel therapeutic approaches. Recent data demonstrated tonic B-cell receptor (BCR) activation of PI3K in BL (Srinivisan et al, Cell 2009); and implicated PI3K, in coordination with MYC, in Burkitt lymphomagenesis (Sander et al, Cancer Cell 2012). In order to investigate mechanisms of resistance, our laboratory generated several resistant BL cell lines. We exposed Raji cells to escalating doses of rituximab +/- human serum and generated several BL rituximab-resistant (Raji 7R and Raji 8RH) (RRCL) or therapy (rituximab-chemotherapy) resistant (Raji 2R and Raji 4RH) (TRCL) cell lines. We then screened for aberrant activation of signal transduction pathways using western blot, phospho-flow cytometry and phosphoproteomics to define pathways associated with the development of resistance. While total Akt expression was similar between all cell lines tested, Raji 2R and Raji 4RH cells had increased phosphorylation of Akt at Ser473 and Thr308, indicating Akt activation, when compared to Raji, Raji 7R and Raji 8RH by western blot and phospho-flow cytometry. Phosphoproteomic analysis comparing Raji and Raji 4RH identified an increase of at least 2 fold in phosphorylation of 315 proteins in Raji 4RH cells, including multiple direct targets of AKT (e.g. GSK3B, WEE1, FOXO1 and PRAS40). Altered phosphorylation of proteins downstream of Akt (e.g. BAD, 4EBP1, GSK3B and ERK) was also detected by western blot in Raji 2R and Raji 4RH compared to Raji, Raji 7R and Raji 8RH. These findings suggest that activation of PI3K/Akt may play a role in the development of chemoresistance noted in our cell line model. KEGG analysis of phosphoproteome and gene expression comparison of Raji to Raji 4RH cells identified the BCR pathway as one of the top pathways altered. Additionally, the microRNA (miR) 17-92 has been noted to increase Akt activation through its inhibitory effects on PTEN, a key regulator of Akt activation. A recent copy number analysis indicated that a recurrent gain in 13q, containing the MIR17HG locus, was also associated with higher mir17 RNA levels and early relapse in pediatric BL patients (Schiffman et al, Br J Haematol 2011). An analysis of miR expression in Raji 2R and Raji 4RH compared to Raji noted a 1.93 and 3.77 fold increase in miR17 respectively. These findings suggest possible mechanisms for increased PI3K/Akt activation in our chemoresistant cell lines. In vitro exposure to MK-2206, a pan-Akt inhibitor, resulted in dose- and time-dependent decreases in viability of TRCLs (IC50 at 72 hours: Raji 2R = 2.6µM; Raji 4RH = 3.2µM) and to a slightly lesser degree Raji and RRCL (Raji= 4.4µM; Raji 7R = 4.0µM; Raji 8RH = 5.2µM) (TRCL vs. Raji/RRCL, p Disclosures Cairo: Celgene: Research Funding.

Introduction: The CD30-positive cutaneous T-cell lymphoproliferative disorders (CD30-positive LPD) include lymphomatoid papulosis (LYP) and primary cutaneous anaplastic large cell lymphoma (ALCL). Recurrent chromosomal translocations frequently underlie the pathogenesis of several hematologic malignancies and often define molecular subtypes with distinct biological behavior. The genetic events that contribute to the pathogenesis of CD30-positive LPD are largely unknown. Goal: The goal of our study was to identify chromosomal translocations that result in the generation of oncogenic chimeric gene fusions that underlie the pathogenesis of CD30-positive LPD. Methods and Results: Paired end whole transcriptome sequencing (RNAseq) analysis was performed on the cutaneous T-cell lymphoma (CTCL) derived cell lines MyLa and HH. Bioinformatic analysis revealed an interchromosomal fusion between NPM1 (5q35) and TYK2 (19p13). The NPM1-TYK2 fusion was represented by 766 paired-end reads spanning the fusion junction of exon 9 of NPM1 and exon 16 of TYK2. Bi-directional Sanger sequencing confirmed predicted gene fusion sequence at the RNA level. SYBR Green I-based quantitative RT-PCR assays revealed specific expression of the NPM1-TYK2 fusion transcript only in MyLa and not in other CTCL (n = 2) or T-cell lymphoma-derived cell lines (n = 7). To establish that the NPM1-TYK2 gene fusion arose from a translocation event at the DNA level, we performed long range PCR which yielded a 1kb product containing the breakpoints and junctions in NPM1 (chr5:170,832,813) and TYK2 (chr19:10,469,817) introns that lead to juxtaposition of these genes by translocation. The fusion is predicted to encode an NPM1-TYK2 protein containing the oligomerization domain of NPM1 and an intact catalytic domain in TYK2. To determine if translocations targeting TYK2 were recurrent, we employed two independent fluorescence in situ hybridization (FISH) based assays, one using a TYK2 break-apart probe strategy to identify translocations targeting TYK2 and a second approach using an NPM1-TYK2 fusion probe strategy to specifically identify the NPM1-TYK2 translocations. These probes were used to screen patient-derived biopsies of CD30-positive LPD, CTCL and other T-cell neoplasms. FISH analyses revealed TYK2 translocations in 5 of 29 (17.2%) primary cases of CD30-positive LPDs (3/15 LYP; 2/14 cutaneous ALCLs) and was absent in other CTCL subtype (Mycosis fungoides, n = 44) and mature T-cell neoplasms (n = 107). Out of these 5 positive cases, one LYP case had an NPM1-TYK2translocation. Since TYK2 is a member of Jak family kinases that are integral components of the JAK-STAT signaling pathway, we investigated the downstream effects of NPM1-TYK2 fusion expression on TYK2 kinase activity and STAT protein pathway activation. Western blotting in MyLa, other cutaneous and mature T-cell lymphoma cell lines revealed hyperactivation of endogenous TYK2 kinase enzyme (pTYK2 levels) only in MyLa. Further, MyLa cells showed activation of downstream STAT signaling pathway proteins (pSTAT1, pSTAT3 and pSTAT5). Ectopic expression of NPM1-TYK2 wild type fusion gene in HEK293FT cells resulted in hyperactivation of TYK2 kinase enzyme and activation of STAT proteins, while a kinase-defective mutant NPM1-TYK2 K462R lacked TYK2 and STAT activity. Transcriptional activation assays for STAT proteins (STAT1, STAT3 and STAT5), showed more than two fold (P Conclusions: Our study demonstrates for the first time recurrent TYK2 gene translocations in CD30-positive LPD or in any form of primary cancer. We identify NPM1 as one of the gene-fusion partners of TYK2 and provide functional support for NPM1-TYK2 in mediating activation of STAT signaling to promote cell proliferation. Inactivation of TYK2 significantly diminishes proliferation, suggesting that TYK2 is an oncogenic driver kinase in a subset of CD30-positive LPD. Finally, our results raise the possibility that TYK2 may be a novel therapeutic target in a subset of CD30-positive LPDs. Disclosures No relevant conflicts of interest to declare.

Background Burkitt Lymphoma (BL) is the most common non-Hodgkin lymphoma (NHL) in children and adolescents and has an excellent prognosis (≥80% 5years, EFS) following short but intense multi-agent chemotherapy (Cairo et al JCO, 2012). The prognosis has improved following the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013). However, a subset of patients with chemoimmunotherapy resistant disease have a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012 and Cairo et al Blood, 2007). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Recently, Czuczman et al have generated a chemoimmunotherapy resistant BL cell line Raji4RH from the native Raji BL line after serial exposures to rituximab (Czuczman et al Clin Cancer Res, 2008). Objective We therefore sought to compare the in-vitro cytotoxicity of rituximab to Raji vs. Raji4RH and the resulting phosphoproteomics changes between untreated and rituximab treated Raji vs. Raji4RH by global phosphoproteomics. Methods In order to identify deregulated signaling proteins/pathways that play a role in BL rituximab resistance, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell line Raji, (CD20+,ATCC, Manhassas, VA) and its rituximab resistant derivative Raji 4RH in the presence or absence of Rituximab (100µg/ml for 24 hr) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides were subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation using a cocktail of 3 anti-phosphotyrosine antibodies. An LTQ Orbitrap XL in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data which were searched with the Swissprot Human taxonomic protein database. Quantitation of identified phosphopeptides was based on spectral counts of phosphorylated peptides. The accuracy of the phospho-site localization was predicted using Luciphor program. Pathway analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Results Our in-vitro results shows that rituximab induced significantly more cell death against Raji vs. Raji4RH (32.7±6.8% vs.5.5±2.2%, p=0.001) compared to isotype control (9.35±0.75% vs.8.9±1.1%, p=NS) at 24 hrs. Furthermore, 2272 unique phosphopeptides were identified and quantified with a probability P> 0.7 and a false localization rate (FLR) < 0.01. These unique phosphopeptides corresponded to 850 unique phosphorylated proteins. Six hundred and nine proteins were differentially phosphorylated (>1.5-fold change) between Raji and Raji 4RH (Figure 1A). Out of these proteins, 374 were more phosphorylated in Raji4RH than in Raji and 235 were more phosphorylated in Raji than in Raji 4RH. Proteins in the spliceosome and the BCR signaling pathway were significantly represented. Changes in the phosphorylation of 430 proteins were induced by Rituximab in Raji cells and involved those in the B-cell receptor (BCR) signaling pathway (LYN, LCK, SLAMF1, PAG1, CD20), the spliceosome (SF3A1, SF3B1, SRSF6), the mTOR pathway (RICTOR, PDPK1, RPS6KA1, BRAF) and the chemokine signaling pathway (CXCR5, PTK2B, CCL1, PRKCD). After rituximab treatment, 559 proteins were differentially phosphorylated between Raji and Raji 4RH (Figure 1B). Rituximab treatment increased the phosphorylation of 262 proteins in Raji 4R while it increased the phosphorylation of 299 proteins in Raji. Conclusions The data indicate that Rituximab results in global changes in proteins in BL involved in not just BCR signaling but also in the spliceosome, the mTOR and the chemokine signaling pathway. Furthermore, aberrant phosphorylation-induced signaling events in the spliceosome and the BCR signaling pathways may contribute to resistance to rituximab. These data revealed the utility of unbiased phosphoproteome interrogation of rituximab mediated signaling events as well as characterizing signaling networks that may provide insights into pathogenetic mechanisms of rituximab resistance. AA and DR considered co-primary first authors, and ML and MC considered co-senior authors. Disclosures: Cairo: Roche/Genentech : advisory board Other.

Treatment with multi-agent chemotherapy regimens has significantly improved survival in pediatric Burkitt Lymphoma (BL) leading to long-term survival in over 80% of cases. The incorporation of rituximab in the treatment of pediatric B-cell non-Hodgkin lymphoma (B-NHL) has been slower than in adults, but may improve clinical outcomes in high risk pediatric BL patients when combined with chemotherapy regimens. On the other hand, BL patients with primary refractory or relapsed disease have a dismal prognosis, stressing the need to identify the mechanism(s) resulting in chemotherapy/rituximab resistance and to develop novel therapeutic approaches. To this end, we exposed a BL cell line (Raji) to escalating doses of rituximab with or without human serum and generated/characterized several BL rituximab-resistant (Raji 7R and Raji 8RH) (RRCL) or rituximab-chemotherapy resistant (Raji 2R and Raji 4RH) (RCRCL) cell lines. Subsequently, we screened for aberrant activation of signal transduction pathways between RSCL (Raji), RRCL (Raji 7R and Raji 8RH), or RCRCL (Raji 2R and Raji 4RH) in an attempt to define what pathways were associated with resistance to both rituximab and chemotherapy agents. This was accomplished by the analysis of phosphorylation patterns on key-regulatory members of pre-defined signal transduction pathways using Western blotting, phospho-flow cytometry studies and phosphoproteomics. Effects on cell viability or cell cycle distribution of RRCL or RCRCL following pharmacological inhibition of key-regulatory pathways identified was then performed using the alamar blue reduction assay or flow cytometry respectively. While total Akt (Protein kinase B) expression was similar between all the types of BL cell lines tested, we found that RCRCL (Raji 2R and Raji 4RH) had an increase in basal phosphorylation levels of Akt at the Ser473 and Thr308 phosphorylation sites when compared to RSCL (Raji cells) or RRCL (Raji 7R and Raji 8RH). These findings were confirmed by phospho-flow cytometry studies. Phosphoproteomic analysis comparing Raji (RSCL) against Raji 4RH (RCRCL) cells identified an increase of at least 2 fold in the phosphorylation of 315 proteins in RCRCL including several direct targets of AKT such as GSK3B, WEE1, FOXO1 and PRAS40. Altered phosphorylation of multiple Akt/mTOR downstream proteins (BAD, 4EBP1, GSK3B and ERK) was detected by western blot in RCRCLs (Raji 2R and Raji 4RH) compared to RSCL and RRCLs. In vitro exposure of RCRCL (Raji 2R and Raji 4RH) to escalating doses of MK-2206, a selective Akt inhibitor, or idelalisib, a selective PI3 kinase delta inhibitor, resulted in a dose- and time-dependent decrease in cell viability of RCRCL and to a lesser degree RRCL and RSCL (RCRCL vs. RRCL/RSCL, p Disclosures: Czuczman: Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

Abstract 896 Anaplastic Large Cell Lymphoma (ALCL) is a common form of pediatric non-Hodgkin lymphoma that is often characterized by the chromosomal translocation t(2;5)(p23;q35) and subsequent expression of the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein. NPM-ALK is a constitutively active tyrosine kinase that functions as a driving oncogenic mutation in ALCL. However, a complete understanding of the signaling mechanisms that contribute to its oncogenesis have not been elucidated. In an effort to identify novel mediators of NPM-ALK signaling, we pursued a phosphoproteomic strategy aimed at the unbiased identification of protein phosphorylation events regulated by NPM-ALK kinase. This analysis resulted in the identification of Pyruvate Kinase M2 (PKM2) as a candidate substrate of NPM-ALK in addition to other metabolic proteins. Phosphorylation of PKM2 is critical for inducing a metabolic shift which leads to accumulation of precursor biomass metabolites required for rapidly dividing cells. We hypothesized that NPM-ALK regulates an oncogenic metabolic shift through phosphorylation of PKM2. Western blot analysis and in vitro kinase assay confirmed that PKM2 is phosphorylated at the Y105 residue by NPM-ALK. The phosphorylation of PKM2 is inversely correlated with its enzymatic activity using an in vitro activity assay. Stable expression of Flag-PKM2 Y105F or treatment with PKM2 small molecule activators (NCGC00186528) results in increased PKM2 activity and induction of a metabolic switch as measured by decreased lactate (80% ± 6%, p Disclosures: No relevant conflicts of interest to declare.

Abstract 583 Background: Reversible phosphorylation is a fundamental post-translational modification which regulates cellular functions by modulation of the activity of critical proteins in cellular signal transduction pathways. Deregulation of protein phosphorylation underlies the pathogenesis of many human diseases, especially cancers. Large scale phosphoproteomic analysis offer an opportunity to catalogue and understand changes in protein phosphorylation associated with cancers and thus provide new possibilities for discovering novel diagnostic biomarkers and new therapeutic targets. Although B-cell non-Hodgkin lymphomas (NHLs) represent the seventh most common cancer in western countries, the extent to which phosphoprotein deregulation may be involved in their pathogenesis is largely unknown. Here we report results of a label-free quantitative phosphoproteomics study of 3 B-cell NHL entities and investigate the contribution of one of the identified protein in the germinal center-derived NHL proliferation. Material and methods: Six milligrams of protein from 13 human B-NHL-derived cell lines (4 mantle cell lymphomas (MCL), 3 Burkitt lymphomas (BL) and 6 follicular lymphomas (FL)) digested by trypsin were subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation using a cocktail of 3 anti-phosphotyrosine antibodies. Phosphopeptides were subjected to liquid chromatography (LC) and MS/MS. Spectra were searched against the UniProtKB database using X!Tandem with k-score. Search results were then post-processed with PeptideProphet and ProteinProphet. All proteins at false discovery rate of 1% were considered for further analysis. Quantitation of identified peptides was based on spectral counts of phosphorylated peptides. Immunoprecipitation and western blot studies were performed to validate the differential phosphorylation of a subset of proteins. The functional consequences of perturbation of an outlier tyrosine phosphorylated protein (PAG1) were explored using shRNA-mediated depletion followed by both colony formation and proliferation assays with or without BCR stimulation by anti-IgM or lipopolysaccharide (LPS). Results: Quantitative phosphoproteomics revealed a total of 7326 serine/threonine phosphorylatided peptides and 392 tyrosine phosphorylated peptides corresponding to 420, 290 and 356 phosphorylated proteins in BL, FL and MCL, respectively. While a subset of phosphoproteins was identified across all lymphoma entities, hierarchical clustering analysis revealed distinctive signatures for all 3 NHL subtypes. Regarding proteins identified with phosphorylated tyrosine residues, BL and FL cell lines were characterized by phosphorylation of proteins implicated in active B-cell receptor signaling such as BTK, LYN, LCK and SYK whereas MCL cell lines were characterized by phosphorylation of proteins implicated in cell cycle control such as CDK1 and CDK2. Interestingly, we identified PAG1, a negative regulator of B-cell receptor signaling, as the most highly tyrosine phosphorylated protein in the germinal center derived cell lines (BL and FL) exclusively. Differential phosphorylation of several proteins was validated by immunoprecipitation and western blot studies. PAG1 silencing in BJAB (BL cell line) by a specific PAG1 shRNA resulted in an increased propensity for colony formation when compared to a scramble shRNA. PAG1 depletion also enhanced significantly the proliferation of BJAB 48h after BCR stimulation by either anti-IgM (3.9-fold increase versus 3-fold increase, p = 0.016) or LPS (4.2-fold increase versus 3-fold increase, p = 0.015). Conclusion: Our study reveals the utility of unbiased phosphoproteome interrogation of B-NHLs to characterize signaling networks that may provide insights into the pathogenetic mechanisms. We identify more than 500 phosphorylated proteins in each NHL entity revealing distinct protein phosphorylation signatures between MCL and germinal center derived lymphomas. Activation of the BCR signaling pathway is characteristic of and pathologically relevant in germinal center derived NHLs. The results of our study reveal novel phosphorylation sites and signatures that may be exploited as diagnostic biomarkers or therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

MCL is a well-defined lymphoid neoplasm characterized by a proliferation of mature B lymphocytes expressing CD5 and with a genetic hallmark, the t(11;14) (q13;q32) translocation leading to the overexpression of cyclin D1, considered as the initial oncogenic event. However this entity may show a spectrum of morphological features broader than initially described. Clinically most of the patients have a poor prognosis with a rapid acquisition of chemoresistance. It has been recently described that some patients may follow an indolent clinical evolution, with the possibility of confusion in diagnosis because of overlapping morphological features with other small B cell lymphomas such as marginal zone lymphoma (MZL) and small lymphocytic lymphoma (SLL). The aim of our study was to analyze patients treated for a B cell lymphoma bearing the t(11;14)(q13;q32) who had a good outcome with a long survival. We retrospectively selected 21 patients (pts) with a t(11;14) B cell lymphoma and who followed an indolent outcome defined by an overall survival of more than 5 years after no more than 1 therapeutic regimen. Feature review included morphologic aspect, immunophenotype, cytogenetics, immunoglobulin (Ig) variable heavy chain (VH) gene usage and mutation patterns. Expression of CD5, CD10, CD23, and CD43, were evaluated on CD19+ cells by FCM. Cytogenetic analysis consisted in conventional cytogenetics on fresh tissue samples completed by a FISH analysis against 11q, 12p, 17p, and 18p realized on fresh frozen material. We further analysed the gene expression profiling of available samples using quantitative real-time PCR using microfluidic cards designed with selected genes of 3 independent signatures (Rosenwald A, et al. Cancer Cell 2003 - Ruiz-Ballesteros E, et al. Blood 2005 - Thieblemont C, et al. Blood 2004). Clinical characteristics showed that median age was 61 (range 45–81). All pts had a good PS and presented with a disseminated disease: bone marrow and blood involvement in 19 (90%) and 11/17 (65%) pts, respectively; peripheral and profound nodes involvement in 15 (71%); and splenomegaly in 13 (62%). LDH and b2-microglobulin levels were elevated in 4 pts. None of the pts had a monoclonal component. IPI score was low in 18 (86%) pts. Splenectomy alone or with chemotherapy (chlorambucil or fludarabine in 4; CHOP in 1) was proposed in 7 pts; monochemotherapy (chlorambucil or fludarabine) in 4 pts, CHOP +/− rituximab (R) in 2 pts, CHOP +/− R followed by autotransplant in 8. One pt did not receive any treatment. With a median follow-up at 6.44 years, median overall survival was 9.18 years. Morphologic review distinguished 2 groups of cases: true MCL cases (MCL, n = 8) and BorderLine Cases (BLC, n=13). Among the 8 MCL and 13 BCL, 7 and 12 were CD5+, 1 and 4 CD5/CD23+, 4 and 5 CD43+, respectively. All cases exhibited a t(11;14), associated with a complex caryotype in 5/8 (62%) MCL and in 9/13 (69%) BLC. Chromosome 7q deletion (n=1) and trisomy chr3/3q (n=3) were present only in BLC cases. IgVH gene was unmutated in 10 cases (3 MCL, 7 BLC) and mutated in 4 (2 MCL, 2 BLC). The most common VH families were VH1 (n=4), VH 3 (n=8) and VH4 (n=3). From the gene expression profiling studies we selected 70 genes specific of the diagnosis of MCL, MZL and SLL. A set of 5 MCL, 5 SLL, and 5 MZL control samples allowed us to validate 25 out of these 70 genes. These 25 genes were further used to analyse MCL and BLC samples. All these samples exhibited a more heterogeneous profile than control samples. Three MCL were more similar to MCL samples, whereas two were closer to MZL samples. All the BLC exhibited a profile close to that of MZL. We further analysed these samples with the 18 gene-signature predicting MCL survival. This signature could not discriminate survival of these MCL and BCL pts. In conclusion, pts with B cell lymphoma bearing the translocation t(11;14)(q13;q32) and with an indolent outcome can be distinguished in at least 2 groups with different characteristics in terms of morphology, immunophenotype, caryotype, and molecular profiles. Using a 25 gene-signature, we found profiles similar to either MCL or MZL for MCL samples, and similar to MZL for BCL samples. The signature discriminating the survival of MCL could not discriminate these indolent MCL. These findings will be validated on a larger series.

Background. Mantle cell lymphoma is one of the most refractory B-cell lymphomas. Despite the recent improvement of novel therapeutic strategies including high dose therapy and the introduction of immunotherapy, MCL patients are not cured by conventional therapeutic strategies. The increasing understanding of the MCL cell biology leads to the development of new drugs targeting to molecular mechanisms of the disease. Among the specific genes found to be overexpressed in MCL by gene expression profiling analysis, farnesyltransferase (FTase) encodes for an enzyme essential in the post- translational modifications of proteins that require prenylation for conversion to mature forms, allowing their participation in various signaling pathways regulating growth and survival. We recently demonstrated that inhibition of FTase by tipifarnib (Zarnestra) is associated in vitro with growth inhibition and apoptosis of MCL cell lines and in vivo with tumor xenograft stability. To determine the efficacy, the safety profile and the toxicity of tipifarnib in MCL, we conducted a phase II trial in patients with refractory MCL, and we evaluated the response considering published molecular predictors. Methods. Primary endpoint was the efficacy measured by the evaluation of the overall response rate (ORR) at 4 cycles, and, in case of response, at 6 cycles. Planned sample size of the study was 27 evaluable patients enrolled for an analysis based on an optimal two-stage design comprising 11 patients for the first stage and 16 for the second stage, under the hypothesis of an ORR of 35% to conclude for an effective drug and an ORR of 10% to conclude for an ineffective drug. Tipifarnib was administered at 300 mg orally twice daily for the first 21 days of each 28-days cycle for 4 to 6 cycles. Prediction of response was retrospectively evaluated in the initial tumor biopsy by the analysis of the 2-gene classifier, the RASGRP1/APTX gene expression ratio, and the AKAP13 expression level. Results. Eleven patients with refractory MCL were included in the analysis. Median number of lines of therapy before tipifarnib was 2.5 (range 1–7). Median age was 71 (range 66–79). All patients presented with stage IV disease and with good performance status. At 4 cycles of tipifarnib, 1 patient presented a complete response and 10 patients were in progressive disease. Two patients progressed during or after the first cycle, three patients after 3 cycles, and five patients after 4 cycles. No grade III-IV hematological toxicities were recorded. One patient presented an unrelated neurological symptom after the first dose administered. Evaluation of the molecular prediction of response to tipifarnib was realized for 3 patients: one responder patient and two non-responder patients. Results showed an increase in the RASGRP1/APTX gene expression ratio and a decrease in AKAP13 expression in the responder patient, and a decrease in the RASGRP1/APTX gene expression ratio and an increase in AKAP13 expression in the non-responder patients. This corresponds to the expected results of the response prediction to tipifarnib. Conclusion. Tipifarnib in refractory MCL was beneficial for only one patient. Response could be exactly predicted by specific molecular predictors of response evaluated in the initial tumor biopsy. These results demonstrate the necessity of categorizing molecularly the patients when targeted therapies are proposed to select those patients that might respond to.