ALBERT

Description: Introduction Hodgkin Lymphoma (HL) accounts for 11% of all lymphomas and despite being one of the most curable lymphomas, 20% of HL patients still ultimately die of their disease. Similarly, a proportion of cases of primary mediastinal B cell lymphoma (PMBCL) have refractory disease or early relapse and frequently fail second-line therapy. Development of more targeted therapeutic approaches is impeded by the lack of knowledge about the mutational landscape in the cancer genomes of these lymphomas. PTPN1 is a protein tyrosine phosphatase gene that encodes the protein, PTP1B. PTP1B dephosphorylates tyrosine residues on many activated kinases to maintain cellular homeostasis. As overactive receptor kinases are critical oncogenic events in cancer, we hypothesized that constitutively active Janus kinase-Signal transducer and activation of transcription (JAK-STAT) observed in HL and PMBCL are in part due to a mutated PTPN1 gene with an impaired functional ability to dephosphorylate this constitutive signaling pathway. Methods and samples Biopsies at the time of primary diagnosis were obtained for 49 PMBCL and 30 HL patients from the British Columbia Cancer Agency, Arizona Lymphoma Repository and the Hôpital Henri Mondor Pathology Department. DNA from PMBCL samples, microdissected Hodgkin Reed Sternberg (HRS) cells and 12 lymphoma-derived cell lines were extracted for PTPN1 exonic PCR amplification (nested PCR was used for HRS cell DNA) and Sanger sequencing. PTPN1 was silenced in a HL cell line (KMH2) by lentiviral transduction of a vector expressing shRNA and confirmed by quantitative real time (qRT) PCR. Wild type and mutant PTPN1 cDNA were cloned into the mammalian expression vector pcDNA 3.1 and expressed in HEK-293 cells. Protein expression of clinical samples, silenced and expressed cells were analyzed by immunohistochemistry and western blotting. Comparisons between groups were performed using two-sample student t tests. Results After exclusion of reported single nucleotide polymorphisms (SNPs) and silent mutations, 16 PTPN1 coding sequence mutations were found in our PMBCL cohort, corresponding to 14 mutations (29%) in clinical samples and 2 in PMBCL-dervied cell lines. Twelve additional mutations were discovered in our HL cohort, corresponding to 6 mutations (20%) in HRS cell samples and another 6 in HL-derived cell lines. In total, 14 (54%) missense, 4 (15%) frameshift, 3 (12%) single amino acid deletions, 4 (15%) nonsense mutations, and 1 (4%) promoter mutation were observed. Eight of these mutations were confirmed as somatic by sequencing of matched constitutional DNA. Silencing of PTPN1 resulted in hyperphosphorylation of JAK1, JAK2, STAT3, STAT5, STAT6 and up-regulation of the oncogenes, MYC and BCL6. Ectopic expression of nonsense and missense PTPN1 mutants in HEK-293 cells led to sustained phosphorylation of STAT6 in comparison to the empty vector control (densitometric values Q9* 0.5 vs. 1.0, R156* 0.7 vs. 1.0, M74L 0.4 vs. 1.0 and M282L 0.8 vs. 1.0). Furthermore, no phosphatase activity was observed for the nonsense mutants and moderate phosphatase activity for the missense mutants using a tyrosine phosphatase-specific substrate (fold change Q9* 2.0, R156* 1.9, M74L 46.7, M282L 46.0 and WT 58.3, compared to empty vector control). Immunohistochemical analysis showed that PTPN1 mutations correspond to decreased protein expression in PMBCL (p=0.03). Discussion PTPN1 is recurrently mutated in PMBCL and HL contributing to constitutive JAK-STAT signaling and oncogene dysregulation. These data suggest PTPN1 mutations as novel driver alterations in these lymphomas and might provide a novel, rational therapeutic target for treating HL and PMBCL patients. Disclosures: Savage: Eli-Lilly: Consultancy. Connors:F Hoffmann-La Roche: Research Funding; Roche Canada: Research Funding.

Description: Abstract 436 Background: Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma that is characterized by the hallmark t(11;14)(q13;q32) translocation, as well as a high number of secondary chromosomal alterations. Further, a small number of genes such as TP53, ATM and CCND1 have been reported to be recurrently mutated in MCL, but do not fully explain the biology and do not adequately account for the wide spectrum of clinical manifestations, response to treatment and prognosis. The aim of this study was to discover new somatic mutations that could contribute to our understanding of the pathogenesis of MCL. Methods: In our discovery cohort, we sequenced the transcriptomes of 18 clinical samples (11 diagnostic and 7 progression biopsies) and 2 mantle cell lymphoma-derived cell lines (Mino and Jeko-1). For this purpose, whole transcriptome shotgun sequencing was performed on RNA extracted from fresh frozen tissue. We assembled an extension cohort of 103 diagnostic patient samples and 4 additional cell lines (Rec-1, Z-138, Maver-1, JVM-2), and performed Sanger sequencing of NOTCH1 exons 26, 27 and 34 on genomic DNA. We further exposed the 6 cell lines to 1 μM of the γ-secretase inhibitor XXI (compound E) for 7 days and measured cellular proliferation with an EdU incorporation assay. Survival analysis was carried out in the 113 patients with diagnostic biopsies and available outcome data. Results: NOTCH1 mutations were found in 14 out of 121 patient samples (11.6%) and in 2 out of 6 cell lines, Mino and Rec-1 (33.3%). The majority of these mutations (12 out of 14) lie in exon 34 that encodes the PEST domain of NOTCH1 and consist of either small frameshift-causing indels (10 cases) or nonsense mutations (2 cases). These mutations are predicted to cause truncations of the C-terminal PEST domain. To gain further insight into functional relevance, we treated 6 cell lines with compound E, an inhibitor of the γ-secretase complex that plays a critical role in the release of the intracellular domain of NOTCH1 after ligand-induced activation. In Rec-1, that harbours a NOTCH1 mutation, we observed a significant decrease in proliferation (mean percentage of cells in culture incorporating EdU decreasing from 47.5% to 1.4%, p

Description: There is proven pre-clinical and clinical efficacy of mono or combinatorial immune strategies to boost host anti-lymphoma immunity, with classical Hodgkin Lymphoma (cHL) seen as the 'poster child'. Approaches include blockade of immune-checkpoints on exhausted tumor-specific T-cells (via mAb blockade of PD-1, TIM3, LAG3, TIGIT or their ligands), activation of T-cells via mAbs agonistic to CD137, and finally modulation of FOXP3, CTLA-4 and/or LAG3 regulatory T-cells (Tregs) or immunosuppressive tumor-associated macrophages (TAMs). In contrast, studies characterizing the circulating and intra-tumoral microenvironment (TME) of the distinct but rare CD20+ Hodgkin Lymphoma entity (5-8% of HL), Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL), are minimal. Furthermore, to our knowledge no functional profiling studies comparing the host immunity of NLPHL with cHL has been performed. We compared host immunity in 29 NLPHL patients, 30 cHL patients and 10 healthy individuals, with a focus on pertinent and clinically actionable immune parameters. Paraffin-embedded tissue and paired (pre- and post-therapy) peripheral blood mononuclear cells samples were interrogated by digital multiplex hybridization (Nanostring Cancer Immune Profiling Panel) and flow cytometry. Although cytotoxic T-cell gene counts (CD8a, CD8b) were similar, compared to cHL there were higher levels of the immune effector activation marker CD137 (gene counts 439 vs. 287; P

Description: Abstract 540 Background: Type 1 diabetes mellitus (T1DM) is a hypercoagulable state associated with increased acute cardiovascular events. Potential risk factors for this include alterations in coagulation and fibrinolytic systems. Tissue factor (TF) is the principal initiator of blood coagulation. Several studies show that there is a circulating pool of TF in blood, which is thrombogenic, and elevated in thrombotic states. We have shown (J Clin Endo Metab 2007, 92:4352-8) that circulating TF procoagulant activity (TF-PCA) is elevated in patients with Type 2 DM (T2DM) and increases further with acute combined hyperglycemia-hyperinsulinemia and selective hyperinsulinemia. There is currently no information on circulating TF-PCA levels and TF responses to hyperglycemia and/or hyperinsulinemia in patients with T1DM who are at comparable risk for cardiovascular events as T2DM patients. Objective: To investigate circulating TF-PCA and other coagulation factors under basal conditions and in response to acute selective hyperglycemia, selective hyperinsulinemia and combined hyperglycemia and hyperinsulinemia in T1DM. Methods: Three study protocols were used: 1) acute correction of hyperglycemia (with IV insulin) followed by 24 h of hyperglycemia, 2) 24 h of selective hyperinsulinemia and 3) 24 h of combined hyperinsulinemia and hyperglycemia. Studies were performed in 9 T1DM patients and 7 non-diabetic subjects. T1DM patients were on a basal/bolus insulin regimen (insulin glargine, 15–70 units at night) or Novolog 70/30 mix twice daily (45-50 units). Circulating membrane bound TF-PCA was measured in whole blood lysates by a two-stage clotting assay (Key et al, Blood; 1998:91). Results: Basal TF-PCA (64.7 ± 6.0 vs. 24.6 ± 1.2 U/ml, p 〈 0.001) and plasma factor VIIa (104 ± 24 vs. 38 ± 8 mU/ml, p 〈 0.03), the activated form of factor VII, were higher in T1DM (n=9) than in non-diabetic controls (n= 7) indicating a chronic procoagulant state. Plasma FVIIc, FVIII, thrombin-antithrombin complexes (TAT) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were not significantly different between patients and controls. When control subjects and T1DM patients were combined, HbA1C correlated with TF-PCA (r=0.71, p=0.0001, n=23). Plasma adiponectin was elevated in T1DM patients compared to control subjects (12.4 ± 1.9 vs 6.7 ± 2.0 μg/ml, p 〈 0.05). Acutely normalizing hyperglycemia in T1DM patients over 3–15 h did not decrease TF-PCA. There were also no changes in plasma FVIIc and FVIII. To explore effects of acutely raising plasma glucose, glucose was raised from 103 ± 8 to ∼ 300 mg/dl by infusion of 20% dextrose and maintained for 24 hours. Insulin concentrations were kept at basal concentrations (6 and 13 μU/ml) by IV infusion. In our previous studies in non-diabetic subjects (Diabetes 2006, 55,202-8) and in T2DM patients (J Clin Endo Metab 2007, 92,4352-8), raising glucose and insulin together produced a marked increase in circulating TF-PCA. We therefore raised glucose to ∼ 250 mg/dl and insulin to ∼ 100 μU/ml together in 8 T1DM patients. Raising glucose levels alone or in combination with insulin decreased circulating TF-PCA by 26% (p 〈 0.02) and 37% (p 〈 0.01), respectively, which is in striking contrast to the elevations noted in non-diabetic controls and T2DM patients. To explore effects of selective hyperinsulinemia, plasma insulin levels were raised in 3 T1DM patients by IV infusion of regular insulin from 15 ± 0.2 to ∼ 75 μU/ml and maintained for 24 hours while plasma glucose was kept at ∼ 100 mg by infusion of 20% dextrose. Again, in contrast to our studies in T2DM patients and healthy subjects we found no increase in TF-PCA Conclusions: Circulating TF-PCA and FVIIa levels are elevated in T1DM patients indicating a potential prothrombotic state. The studies on acutely induced hyperinsulinemia and hyperglycemia indicate that the regulation of TF expression is different in T1DM and T2DM. This may be due to multiple mechanisms, including a differential effect of insulin on monocytes TF expression in T1DM and T2DM, and due to differences in plasma adiponectin, which has been shown to inhibit TF expression and is elevated in T1DM. Additional studies are needed to obtain insights into the mechanisms regulating the differential expression of TF in the two forms of diabetes. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Somatic IL4R mutations were identified in 24% of primary PMBCL cases (n = 62) and in 100% of PMBCL-derived cell lines. IL4R mutations lead to hyperphosphorylation of STAT proteins activating downstream immunoregulatory genes (CD23, CCL17).

Description: Key Points Programmed death ligands 1 and 2 are rearranged at a frequency of 20% in PMBCL.

Description: In classical Hodgkin lymphoma (CHL) the tumor microenvironment (TME) is enriched in T cells that modulate antitumor immunity. PD1 blockade partially restores anti-tumoral T cell function, to induce impressive responses in a proportion of patients with relapsed/refractory CHL (Chen et al JCO 2017). Further characterisation of T cell immune evasion mechanisms in CHL will permit the rational development of enhanced immunotherapeutic strategies. Lymphocyte-activation gene 3 (LAG3) is a cell surface molecule known to be expressed on a subset of immune effector T cells and intratumoral regulatory T cells (Tregs) in solid-organ tumors, with combination PD1/LAG3 mAb blockade showing early promise (Ascierto et al 2017 JCO abst 9520). In contrast, data in haematological malignancies is limited, although it is known that LAG3+ T cells suppress anti-tumoral immunity in CHL and B-CLL (Gandhi et al Blood 2006; Shapiro et al Haematologica 2017). Interestingly, in B-CLL LAG3 is found on both T cells and malignant B cells. Whether Hodgkin Reed-Sternberg (HRS) cells express LAG3 is unknown. To characterise in detail intratumoral and circulating LAG3 in CHL we used a conventional discovery / validation approach. The local institutional discovery cohort was drawn from Princess Alexandra Hospital in Brisbane (Australia), and validated in samples from the prospective randomised phase III international "RATHL" trial (Johnson et al NEJM 2016). Firstly, LAG3 gene expression (GEP) was digitally quantified by NanoString in FFPE tissues in the discovery cohort and compared to normal control nodes and DLBCL tissues. Normalised LAG3 mRNA counts were 5-10-fold higher in CHL than in controls (P 〈 0.001) and 3-5-fold higher than in DLBCL (P 〈 0.001), whereas PD1 and TIM3 mRNA counts did not differ. In CHL samples LAG3 mRNA counts were markedly increased compared to PD-1 axis molecules and TIM3 (P 〈 0.001) (Figure 1a). Higher levels of LAG3 mRNA counts were correlated with infiltration by T cells (CD4 r = 0.55; P 〈 0.00001; CD8 r = 0.51, P 〈 0.0001), and macrophages (CD68 r = 0.45; P = 0.002). Findings were replicated in the RATHL cohort. Next, intratumoral LAG3 cellular distribution was established. Flow cytometry was used to quantify LAG3 in T cell subsets and CD30+CD3- HRS cells in 6 de-aggregated freshly frozen CHL nodes (TILs). LAG3 was evenly distributed between CD8+ T cells, CD127LOCD25HI natural-Tregs (nTregs) and CD127LOCD25LO induced-Tregs (iTregs), but with minimal expression on CD4 non-Tregs, with the latter constituting the majority of intratumoral T cells. LAG3+ T cells typically co-expressed PD1 and/or TIM3. LAG3 was expressed on CD30+CD3+ cells but not on CD30+CD3- cells, consistent with LAG3 expression on activated T cells. Multispectral immunofluorescence (mIF) image analysis confirmed these findings in histological tumor samples (Figure 1b). Also, there was negligible expression of LAG3 on HRS-lines. Finally, the potential role of soluble LAG3 (sLAG3) as a rapid-turnaround circulating biomarker applicable to the routine diagnostic laboratory, was assessed in serum samples using the MSD R-PLEX assay. In the discovery cohort sLAG3 was 3-4-fold increased at pre-therapy compared to controls and 3-6M post-therapy serum (P = 0.001). Results from pre-therapy RATHL serum samples were similar (P 〈 0.05). Notably in RATHL samples at interim restaging after 2 ABVD cycles sLAG3 had reduced by ~5-fold compared to pre-therapy (P 〈 0.0001) in patients with PET/CT responsive disease (Figures 1 c + d). Twelve months post therapy sLAG remained significantly lower than pre-therapy (P 〈 0.05) and was equivalent to control samples. Pre-therapy serum sLAG3 demonstrated a modest correlation with tissue LAG3 mRNA counts (r = 0.45; P = 0.02). In conclusion in CHL, LAG3 mRNA expression was markedly increased relative to control and DLBCL tissues. Within CHL tissues LAG3 mRNA was markedly increased compared to other immune checkpoint molecules. Interrogation of the TME using flow cytometry of TILs and mIF demonstrated LAG3 is evenly distributed between CD8+, nTregs and iTRreg. In tumor samples we did not find evidence of LAG3 expression on HRS cells. To our knowledge this is the first study to demonstrate sLAG3 as a cell free circulating disease response biomarker in CHL. Taken together these findings provide a convincing rationale for further exploration of single and/or combined checkpoint blockade incorporating LAG3 inhibition to treat CHL. Disclosures Abro: Bristol-Myers Squibb: Speakers Bureau; Janssen: Other: education support congress attendance; Celgene: Other: education support congress attendance; Novartis: Consultancy; Amgen: Other: education support congress attendance. Keane:BMS: Research Funding; Roche: Other: Education Support, Speakers Bureau; Celgene: Consultancy, Research Funding; Takeda: Other: Educational Meeting; Merck: Consultancy. Birch:Medadvance: Equity Ownership. Tobin:Amgen: Other: Educational Travel; Celgene: Research Funding. Johnson:Kite: Consultancy; Celgene: Honoraria; Eisai: Research Funding; Incyte: Consultancy; Takeda: Honoraria, Travel, accommodations, expenses; Janssen: Consultancy, Research Funding; Genmab: Consultancy; Novartis: Honoraria; Zenyaku Kogyo: Other: Travel, accommodations, expenses; Bristol-Myers Squibb: Honoraria; Boeringher Ingelheim: Consultancy; Epizyme: Consultancy, Honoraria, Research Funding. Trotman:F. Hoffman-La Roche: Other: Travel to meeting, Unremunerated member of Ad Board, Research Funding; PCYC: Research Funding; Janssen: Other: Unremunerated member of Ad Board, Research Funding; Takeda: Other: Unremunerated member of Ad Board; Celgene: Other: Unremunerated member of Ad Board, Research Funding; Beigene: Research Funding. Bird:Amgen, Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Gill:Janssen: Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau. Gandhi:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Takeda: Honoraria; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Background The frequent loss of Major Histocompatibility Complex I molecule (MHC-I) on Hodgkin/Reed-Sternberg (HRS)-cells renders them susceptible to Natural Killer (NK) cell-mediated lysis in Hodgkin Lymphoma (HL). Optimal NK cell function involves migration from peripheral blood to sites of disease, formation of an immune synapse (NKIS) between NK cells and HRS cells, and release of effector molecules. We recently showed that PD-1+ NK cells are expanded in the circulation of patients with HL (Vari, F Blood 2018), and that PD-1 blockade enhances their anti-HRS capabilities. However, mechanisms behind the functional impairment of NK cells in HL patients and the impact PD-1 blockade has on NK cell function remain to be established. Although the IRE1-XBP1s pathway, part of the unfolded protein response (UPR) system, has established and fundamental roles in macrophage, B, T and dendritic cells homeostatic function, its involvement in NK cells remains unknown. We hypothesized that IRE1-XBP1s dysfunction contributes to NK cell impairment and tested the impact of PD-1 blockade on individual components of NK cell function, including migration, NKIS formation, and cytokine release. Methods Ex-vivo functional assays were performed on blood from 20 participants. Confocal microscopy, time-lapse imaging, trans-well migration, and functional in-vitro immune assays were utilized on a range of NK and HRS cell lines, with and without IRE1-XBP1s small molecule inhibitors (4µ8c and 6-bromo) and/or PD-1 blockade (pembrolizumab). Results Stimulation of both NK cell lines and primary NK cells, with HRS lines resulted in marked and rapid IRE1-XBP1s pathway activation. This occurred independently of the canonical UPR and was associated with increased NK cell effector function. However, IRE1-XBP1s pathway inhibition resulted in aberrant NK cell morphology, reduced motility and migration, deficient NKIS formation and impaired interferon-gamma (IFNγ) and tumor necrosis factor alpha (TNFα) release. Next, we tested the IRE1-XBP1s pathway in the pre-therapy blood of patients with HL and compared this with healthy age/gender matched controls. Strikingly, following co-culture with an HRS-line the pathway was not activated, but this abnormality was restricted to the CD56brightCD16-ve subset that we have previously shown to be expanded and enriched in PD-1 (as well as downregulation of the lymphoid migratory chemokine CCR7) in patients with HL. In subsequent experiments using in-vitro expanded populations of primary NK cells from HL patients, IRE1-XBP1s pathway inhibition impaired the migration, NKIS formation, CD107a degranulation, and secretion of IFNγ and TNFα. Effects were partially but not completely restored by addition of PD-1 blockade (Fig 1). Conclusion Here, we outline a hitherto unrecognized mechanism involving the IRE1-XBP1s pathway that is pivotal to NK cell function, including the relatively poorly understood processes of migration, NKIS formation, and cytokine secretion. Notably, IRE1-XBP1s pathway activation is dysfunctional within the PD-1 enriched CD56brightCD16- NK cell subset. Although PD-1 blockade appears to have a multi-faceted beneficial role on NK cell migrational/NKIS and cytokine release capabilities, it is still only capable of partial restoration of NK cell effector function. Further understanding of the pathways operative in NK cells may result in improved immunotherapeutic strategies to enhance this arm of the immune response in patients with HL. Disclosures Gandhi: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding.