ALBERT

Description: Introduction: Castleman disease (CD) is an uncommon lymphoproliferative disorder of unclear etiology. CD is broadly subclassified into unicentric (UCD) and multicentric (MCD) disease based on based on clinical, radiological, laboratory and histomorphological findings. UCD presents with minimal symptoms, is restricted to a single lymph node station, and excision is usually curative. Multicentric CD, in contrast, presents with systemic inflammatory symptoms, multicentric lymphadenopathy, and vital organ dysfunction. MCD is further subclassified into Human Herpesvirus-8(HHV-8)-associated MCD and HHV-8-negative/idiopathic MCD (iMCD), the etiology of which is poorly understood. Histologically, UCD frequently shows a hyaline vascular pattern with atretic follicles, thickened mantle zones and increased interfollicular vascularity with hyalinization. iMCD frequently shows increased interfollicular plasma cells and hyperplastic follicles. iMCD is also characterized by a systemic hypercytokinemia that includes IL-6, VEGF, IL-2, TNF-α, IL-10 and CXCL13. However, the cell(s) involved in initiating and amplifying the cytokine network are unclear. We combined histomorphology and cytokine in situ hybridization to identify the hypercytokine-producing cells in lymph nodes from CD patients. In addition, T and B lymphocytes play important roles in initiating and amplifying the cytokine response and their clonality has not been well-studied in CD. Here, we performed deep sequencing of the immunoglobulin heavy chain and T cell receptor gene loci. Methods: Lymph node biopsies from patients with UCD and iMCD were identified from the pathology archives of the Children's Hospital of Philadelphia. 17 UCD cases and 8 HHV8-negative iMCD cases were examined. Reactive lymph nodes (N=10) with plasmacytosis and/or other CD-like features served as controls. Cytokine expression was determined by RNA in situ hybridization (RNAscope) on formalin fixed paraffin embedded tissue. IL-6, IL-6R, VEGF, IL-10, TNF-α, IL-1β, IL-2, and IL-8 RNA expression patterns were analyzed in conjunction with histomorphological features. Expression was manually quantified with a semi-quantitative grading scale (0-4) per manufacturer recommendations and statistical analysis was performed using the Chi-square test. Fresh frozen lymph node tissue was utilized for deep sequencing of the TCR Vβ and IgH gene loci. VDJ usage, clonal frequency and CDR3 sequence was determined and compared between subtypes of CD and reactive lymph node controls. Results: Lymph nodes from patients with iMCD express significantly higher levels of VEGF compared to patients with UCD and controls (75% vs. 29% vs. 0%; p=0.014). Atretic follicles and interfollicular regions were the source of increased VEGF expression. Potential cell types responsible for the increased VEGF production in these regions are follicular dendritic cells in the atretic follicles and plasma cells in the interfollicular areas. IL-6 expression was also significantly higher in iMCD cases compared to UCD and controls (75% vs. 25% vs. 20%, p=0.026) in a subset of cells within the interfollicular regions. This cellular source of the excess IL-6 in the interfollicular region may be endothelial cells. Thus, follicular dendritic cells in the germinal centers and endothelial cells, T cells, and plasma cells in the interfollicular spaces are potential sources for increased VEGF and IL-6. IL-6R, IL-10, TNF-α, IL-1β, IL-2, and IL-8 showed no significant differences between the various subtypes of CD. Deep sequencing of the TCRα gene loci revealed mildly expanded clonal T-cell populations (5% of total sequences) in a subset of iMCD cases (2/6) and UCD cases (1/9) compared to controls (0/15). B cell populations were polyclonal in both subtypes of CD and in reactive lymph nodes. Conclusion: The findings suggest that cells in the interfollicular region and atretic follicles in the lymph nodes are a potential source of the systemic hypercytokinemia in iMCD. The locations and patterns of cytokine expression implicate follicular dendritic cells, endothelial cells, and plasma cells specifically as potential hypercytokine-producing cells. Additionally, T cells in CD show oligoclonality in some cases and may play an important role in initiating or amplifying the immune response in CD. Disclosures Fajgenbaum: Janssen Pharmaceuticals, Inc.: Research Funding.

Description: Castleman disease (CD) describes a group of heterogeneous diseases defined by shared characteristic lymph node histopathology and is classified based on the number of regions of enlarged lymph nodes. Multicentric CD (MCD) involves multiple regions of lymphadenopathy as well as systemic inflammation, cytopenias, and vital organ dysfunction due to a cytokine storm that often includes interleukin-6. In ~50% of patients, the pathogenic driver is Kaposi sarcoma-associated/human herpesvirus-8 (HHV-8) in the context of immunosuppression. In contrast, the etiologic driver in HHV8-negative MCD (idiopathic or iMCD) is unknown. To date, most research has focused on descriptive characterization of the enlarged lymph nodes, and the pathological cell types driving iMCD pathogenesis remain unidentified. Given that lymphoid cells circulate through the blood and lymph nodes, are able to produce high levels of cytokines upon activation, and are the primary cell types responsible for the enlarged lymph nodes in iMCD and other related diseases, we first performed a detailed immunophenotyping of peripheral blood mononuclear cells (PBMCs) obtained from iMCD patients in remission (n=16), iMCD patients during disease flare (n=6) and healthy donors (HD) (n=15). PBMCs were isolated by density gradient and either stained immediately or cryopreserved for future analyses. A HD sample was drawn at the same time as each experimental sample and processed and analyzed in parallel. Our initial hypothesis was that analysis of iMCD flare PBMCs would reveal an abnormal myeloid or lymphocyte subset. Thus, we stained and analyzed PBMCs for standard lineage markers: CD11b, CD15, CD19, CD3, CD56 and CD14. However, we observed no gross differences in population frequencies during either remission or flare compared to HD. Additionally, no differences in the proportions of natural killer T cells (CD3+CD56+), or CD4+ or CD8+ lymphocytes were observed. However, more refined examinations of the lymphocyte sub-sets based upon activation status revealed an increased proportion of activated memory (CD62LlowHLA-DR+) CD8+ cells during iMCD flare compared to HD and iMCD patients in remission and a decreased proportion of naïve (CD62L+CD45RA+) CD8+ cells compared to HD (p

Description: While human B cells have been extensively studied, most reports have used peripheral blood as a source. Here we have used a unique tissue resource derived from healthy organ donors to deeply characterize human B cell compartments across multiple tissues and donors. These data sets revealed that B cells in the blood are not in homeostasis with compartments in other tissues. We found striking donor-to-donor variability in the frequencies and isotype of CD27+ memory B cells (MBC). A comprehensive Ab-based screen revealed markers of MBC and allowed identification of novel MBC subsets with distinct function defined by surface expression of CD69 and CD45RB. We defined a tissue-resident MBC phenotype that was predominant in the gut but absent in blood. RNA sequencing of MBC subsets from multiple tissues revealed a tissue-resident MBC gene signature as well as gut and spleen-specific signatures. Overall, these studies provide novel insights into the nature and function of human B cell compartments across multiple tissues.

Description: Prevailing approaches to manage autoimmune thrombotic disorders, such as heparin-induced thrombocytopenia, antiphospholipid syndrome and thrombotic thrombocytopenic purpura, include immunosuppression and systemic anticoagulation, though neither provides optimal outcome for many patients. A different approach is suggested by the concurrence of autoantibodies and their antigenic targets in the absence of clinical disease, such as platelet factor 4 in heparin-induced thrombocytopenia and β2-glycoprotein-I (β2GPI) in antiphospholipid syndrome. The presence of autoantibodies in the absence of disease suggests that conformational changes or other alterations in endogenous protein autoantigens are required for recognition by pathogenic autoantibodies. In thrombotic thrombocytopenic purpura, the clinical impact of ADAMTS13 deficiency caused by autoantibodies likely depends on the balance between residual antigen, that is, enzyme activity, and demand imposed by local genesis of ultralarge multimers of von Willebrand factor. A corollary of these concepts is that disrupting platelet factor 4 and β2GPI conformation (or ultralarge multimer of von Willebrand factor oligomerization or function) might provide a disease-targeted approach to prevent thrombosis without systemic anticoagulation or immunosuppression. Validation of this approach requires a deeper understanding of how seemingly normal host proteins become antigenic or undergo changes that increase antibody avidity, and how they can be altered to retain adaptive functions while shedding epitopes prone to elicit harmful autoimmunity.

Description: Introduction: Preclinical data suggest that PD-1 and PD-L1/PD-L2 mediate immune evasion in CLL. However, clinical data (Ding, BLOOD 2017) demonstrate that pembrolizumab monotherapy (pembro) is ineffective in relapsed/refractory (r/r) CLL patients (pts) (ORR 0%, med PFS 2.4 months). Additional data with an anti-PD1 demonstrated a 90% failure rate in Richter's Transformation (RT) with a med 2-month OS (Rogers, BJH 2018). Combinations may represent the future of anti-PD-1 therapy in CLL/RT, especially in RT pts rel/ref to ibrutinib, where median OS is ~3.5 mos. We hypothesized synergistic activity with PD-1 + PI3K blockade. Umbralisib (UMB), is a highly-specific PI3K-δ inhibitor with additional effects on casein kinase-1 epsilon (CK-1ε), which may have an inhibitory effect on Treg function (Deng et al, 2016). We tested the safety and activity of UMB in combination with the anti-CD20 mAb ublituximab (UTX) and pembro in r/r CLL and RT - the first reported combination of a PD-1 inhibitor + PI3K-δ inhibitor in this population. Methods: Ph I (3+3 design), multicenter study to assess the safety/efficacy of UMB + UTX + pembro in pts with r/r CLL and RT. Treatment for CLL pts involved three stages: Induction: UMB (800mg daily) and UTX (900mg 3 out of 4 wks) for the first two 28-day cycles. Consolidation: Pembro (dose level 1=100mg, dose level 2=200mg) was then initiated every 3 wks in combination with UMB and UTX (900mg week 2 of cycle 4 and 6) for cycles 3-6. Maintenance: Cycle 〉 6, UMB 800 mg daily until progressive disease (PD) or unacceptable AE. For RT pts, all study drugs are started in cycle 1 with the following schedule: UMB 800 mg daily; UTX 900 mg cycle 1 (D1, 8, 15), D1 cycles 2-4, cycle 7 and q3 cycles thereafter; and pembro D3 of cycle 1 and D2 of cycles 2-4. The primary endpoint is safety of the triple combination with efficacy as a secondary endpoint. Response assessments for CLL, based on the iwCLL 2008 criteria, were performed after cycles 2, 6 and 12. RT response assessments (Cheson 2007) were completed at the end of cycles 2, 4 and every 3 cycles until month 12. Peripheral blood and/or bone marrow biopsy, enriched for mononuclear cells, were obtained for correlative analyses at screening, month 2 and 6. Results: Fourteen pts have been treated to date: 9 with CLL (3 at 100 mg pembro / 6 at 200 mg pembro) and 5 with RT (4 at 100 mg pembro / 1 at 200 mg). Baseline demographics were as follows: male/female (9/5), med age 71 yrs (range 60-81), med prior therapies 2 (1-8), 79% were refractory to immediate prior therapy. 10 pts had prior ibrutinib of which 9 were ibrutinib refractory. 64% of pts had at least 1 high risk genetic feature (del17p, del11q, TP53 mut, NOTCH1 mut or complex karyotype). AE's (all causality) were manageable. Grade 3/4 AE's occurring in ≥ 3 pts included neutropenia (n=6, 43%), ALT/AST increase (n=3, 21%) and hypophosphatemia (n=3, 21%). One DLT occurred in a CLL pt at 200 mg dose level (ALT/AST elevation) which trigged expansion to 6 pts. No additional DLTs were reported, and no MTD was achieved. No increase in expected grade ≥ 3 PI3Kδ-associated toxicities were noted (1 pneumonitis event, no colitis events). ORR was 89% for CLL pts with ORR of 75% in BTK refractory CLL pts (3/4). Notably 2/4 BTK refractory CLL pts achieved a response to UMB + UTX (U2) induction alone prior to the addition of pembro. 4/5 RT pts were eligible for efficacy evaluation with 50% ORR (2/4, CR). The 2 RT CRs were durable and are ongoing (15+ mos and 7+ mos); both pts were ibrutinib refractory and had 7 (including SCT) and 8 prior lines respectively, and one had failed CAR-T. With a med follow up of 15 mos, 10/14 (71%) remain progression free (range 3 - 44 mos, Fig 1), including one CLL pt who completed consolidation and has been off therapy for 27+ mos. Sequential sampling for correlative studies demonstrated modest changes in Tregs numbers (Fig 2). Conclusion: The triplet combination of umbralisib + ublituximab + pembro was well-tolerated. Responses were durable in BTK refractory, high risk pts, including two CR's in RT pts. In contrast to pembro monotherapy, data suggest that CLL/RT pts who achieve ≥ PR with this checkpoint inhibitor-containing regimen can achieve durable responses. Correlative studies suggest that maintenance of Tregs may limit autoimmune sequelae. Enrollment is ongoing in both the CLL (BTK refractory only) and RT cohorts. Disclosures Mato: AbbVie: Consultancy, Research Funding; Portola: Research Funding; Regeneron: Research Funding; AstraZeneca: Consultancy; Johnson & Johnson: Consultancy; Celgene: Consultancy; Medscape: Honoraria; TG Therapeutics: Consultancy, Research Funding; Pharmacyclics, an AbbVie Company: Consultancy, Research Funding; Acerta: Research Funding; Prime Oncology: Honoraria. Svoboda:Regeneron: Research Funding; KITE: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; TG Therapeutics: Research Funding; Pharmacyclics: Consultancy, Research Funding; Kyowa: Consultancy; Seattle Genetics: Consultancy, Research Funding; Merck: Research Funding. Schuster:Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Research Funding; Dava Oncology: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Honoraria, Research Funding. Becker:GlycoMimetics: Research Funding. Brander:DTRM: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; BeiGene: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Novartis: Consultancy, Other: DSMB; Pharmacyclics, an AbbVie Company: Consultancy, Honoraria, Research Funding; Acerta: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; TG Therapeutics: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Teva: Consultancy, Honoraria; Genentech: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; AbbVie: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding. Dwivedy Nasta:Aileron: Research Funding; Pharmacyclics: Research Funding; Debiopharm: Research Funding; Incyte: Research Funding; Rafael/WF: Research Funding; Roche: Research Funding; Takeda/Millenium: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Merck: Other: DSMC. Landsburg:Takeda: Consultancy; Curis: Consultancy, Research Funding. Kennard:AbbVie, Gilead, Verastem: Consultancy. Zelenetz:Abbvie: Research Funding; Celgene: Consultancy; AstraZeneca: Consultancy; Novartis/Sandoz: Consultancy; Amgen: Consultancy; Gilead: Consultancy, Research Funding; Genentech/Roche: Consultancy, Research Funding. Purdom:TG Therapeutics: Employment, Equity Ownership. Paskalis:TG Therapeutics: Employment, Equity Ownership. Sportelli:TG Therapeutics: Employment, Equity Ownership. Miskin:TG Therapeutics: Employment, Equity Ownership. Weiss:TG Therapeutics: Employment, Equity Ownership. Shadman:Gilead Sciences: Research Funding; Celgene: Research Funding; AstraZeneca: Consultancy; Beigene: Research Funding; AbbVie: Consultancy; Genentech: Research Funding; Qilu Puget Sound Biotherapeutics: Consultancy; Genentech: Consultancy; Mustang Biopharma: Research Funding; Acerta Pharma: Research Funding; TG Therapeutics: Research Funding; Pharmacyclics: Research Funding; Verastem: Consultancy.