Abstract: Chronic obstructive pulmonary disease (COPD) is associated with age and smoking, but other determinants of the disease are incompletely understood. Clonal hematopoiesis of indeterminate potential (CHIP) is a common, age-related state in which somatic mutations in clonal blood populations induce aberrant inflammatory responses. Patients with CHIP have an elevated risk for cardiovascular disease, but the association of CHIP with COPD remains unclear. We analyzed whole-genome sequencing and whole-exome sequencing data to detect CHIP in 48 835 patients, of whom 8444 had moderate to very severe COPD, from four separate cohorts with COPD phenotyping and smoking history. We measured emphysema in murine models in which Tet2 was deleted in hematopoietic cells. In the COPDGene cohort, individuals with CHIP had risks of moderate-to-severe, severe, or very severe COPD that were 1.6 (adjusted 95% confidence interval [CI], 1.1-2.2) and 2.2 (adjusted 95% CI, 1.5-3.2) times greater than those for noncarriers. These findings were consistently observed in three additional cohorts and meta-analyses of all patients. CHIP was also associated with decreased FEV1% predicted in the COPDGene cohort (mean between-group differences, −5.7%; adjusted 95% CI, −8.8% to −2.6%), a finding replicated in additional cohorts. Smoke exposure was associated with a small but significant increased risk of having CHIP (odds ratio, 1.03 per 10 pack-years; 95% CI, 1.01-1.05 per 10 pack-years) in the meta-analysis of all patients. Inactivation of Tet2 in mouse hematopoietic cells exacerbated the development of emphysema and inflammation in models of cigarette smoke exposure. Somatic mutations in blood cells are associated with the development and severity of COPD, independent of age and cumulative smoke exposure.

Abstract: We have previously reported that selection of marrow cells on the basis of the CD34+HLA-DR- phenotype (34+DR-) may result in the recovery of Philadelphia chromosome (Ph)- and BCR/ABL-negative long-term culture- initiating cells (LTC-IC) in selected patients with chronic myelogenous leukemia (CML). We now present data on 27 early chronic-phase ([ECP] studied within 1 year after diagnosis) and 23 advanced-phase ([AP] late chronic phase, ie, studied 〉 1 year from diagnosis, or accelerated phase) CML patients. Fluorescence-activated call-sorting (FACS)- selected 34+DR- and 34+DR+ cells were subjected to reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization. These cells were also cultured in long-term bone marrow culture for 1 to 5 weeks to examine the number of LTC-IC and the presence or absence of the BCR/ABL gene rearrangement in progeny of primitive LTC-IC. The number of 34+DR- cells and LTC-IC present in ECP CML marrow was similar to that in normal (NL) marrow, whereas the numbers were reduced in AP CML. Furthermore, 34+DR- cells from more than 80% of ECP CML patients were BCR/ABL mRNA- and Ph-negative and contained only BCR/ABL mRNA- and Ph-negative LTC-IC, whereas 34+DR- cells and LTC-IC from less than 40% of AP CML patients were BCR/ABL mRNA- and Ph-negative. In contrast to NL marrow, 34+DR+ cells from CML marrow, irrespective of clinical stage, contained large numbers of LTC- IC. CML 34+DR+ cells and LTC-IC were BCR/ABL mRNA- and Ph-positive. Since these studies suggested that a population of primitive progenitors that are Ph-negative can be selected from steady-state marrow in some ECP CML patients, we determined if similar results could be obtained when large quantities of marrow sufficient for transplantation are processed. We demonstrate that 1 to 3 x 10(5) BCR/ABL mRNA-negative 34+DR- cells/kg recipient body weight, containing only BCR/ABL mRNA-negative LTC-IC, can be obtained from a 2- to 2.5-L marrow collection by sequential COBE Spectra apheresis (COBE BCT, Lakewood, CO), CD34+ enrichment using the CEPRATE SC Cell-Concentrator (CellPro, Bothell, WA), and high-speed FACS. Thus, large-scale selection of a BCR/ABL mRNA- and Ph-negative 34+DR- cell population is possible in a fraction of chronic-phase CML patients, in whom these cells could be used to reconstitute the hematopoietic compartment following autologous transplantation.

Abstract: Single-agent IL-15/IL-15Rα-Fc (ALT-803) therapy was well tolerated and resulted in clinical responses in patients who relapsed post-HCT. First-in-human use of ALT-803 promoted NK and CD8+ T-cell expansion and activation in vivo without stimulating regulatory T cells.

Abstract: The PD-1/PD-L1 pathway plays an important role in regulation of alloimmune responses and in induction and maintenance of peripheral tolerance. Because GVHD is driven by donor T cells and PD-L1 expression can be markedly elevated on T cells during activation, we investigated the functional significance of PD-L1 expressed by donor T cells in regulating murine models of acute GVHD. PD-L1 expression was up-regulated on donor CD4 and CD8 T cells during GVHD. We considered the possibility that PD-L1 expression on activated donor T cells might inhibit GVHD by down regulating donor anti-host T cell responses, consistent with PD-L1 co-inhibitory activity when expressed on host parenchymal cells during GVHD. Surprisingly, T cell mediated GVHD lethality was markedly reduced in recipients of PD-L1-/- compared to WT donor T cells in both B6 to BALB/c model of GVHD(P 〈 0.0001; Fig 1A) and in B6 to B10.BR model (P=0.0047; Fig 1B), suggesting that PD-L1 expression on donor T cells is involved in interactions that enhance T cell mediated effector function. Survival data confirmed that PD-L1-/- Teffs and not Tregs were responsible for reduced lethality in recipients of PD-L1-/- donor T cells (Fig 1C). During GVHD, PD-L1-/- donor CD4 and CD8 T cells had reduced expression of gut homing receptors (Fig 1D), and recipients of PD-L1-/- donor T cells had reduced T cell infiltration into lymphoid organs and gut, retained intestinal epithelial integrity, and had lower inflammatory cytokine production. PD-L1-/- donor CD4 and CD8 T cells had increased expression of multiple inhibitory receptors (Fig 1E, 1F), reduced T cell proliferation, and increased T cell apoptosis by transcriptional profiling and cell surface marker expression. Four pathways, including proteasome activity showed decreased expression in PD-L1-/- donor T cells. In vitro T cell activation in the presence of single (PD-L1:B7-1) vs. dual (PD-L1:B7-1 and PD-L1:PD-1) pathway blocking anti-PD-L1 mAb confirmed that T-T interaction between PD-1 and PD-L1 is important for proliferation and survival, whereas sensitive in vitro assays with supported lipid bilayers found no evidence for a functionally relevant cis interaction of PD-L1 and PD-1 on T cells. We found a significant increase in glucose transporter (GLUT1) expression in proliferating WT vs. PD-L1-/- donor CD4 and CD8 T cells, along with increased glycolysis, OXPHOS, glutamine consumption and glutamate production. We also observed increased fatty acid (FA) uptake and FA oxidation, and enhanced pharmacologic inhibition of FA oxidation in WT donor T cells, suggesting that PD-1:PD-L1 interactions are important for FA metabolism, which may further support T cell survival. Studies using stable isotope carbon tracers highlighted the divergent roles for glutamine and glucose in energy generation and biosynthetic pathways. Given the importance of acetyl-CoA as a high energy thioester intermediate in the TCA cycle and a lipogenic precursor for T cells undergoing expansion, significantly enhanced production of acetyl-CoA from glucose by WT donor T cells support the notion that PD-L1 on T cells promotes clonal expansion of alloreactive T cells. In summary, these data are the first to show that PD-L1 expression on donor T cells can provide positive signals for T cell survival, activation, and metabolism. Greater understanding of the function of PD-L1 expression by activated donor T cells will provide new insight into the regulation of GVHD and suggest strategies to selectively inhibit PD-L1 on donor T cells that may be clinically useful to prevent GVHD. Figure 1. PD-L1-/- vs. WT donor T cells lessen GVHD lethality, independent of donor Treg function. (A) Survival of BALB/c mice with WT B6 or PD-L1-/- T cells. (B) Survival of B10.BR mice with WT B6 or PD-L1-/- T cells. (C) Survival of BALB/c mice with WT B6 or PD-L1-/- T cells, or with WT B6 or PD-L1-/- CD25 depleted T cells (recipients of WT T cells vs. WT CD25-depleted T cells, P = 0.0003; recipients of PD-L1-/- T cells vs. PD-L1-/- CD25-depleted T cells, P = 0.2306; recipients of WT vs. PD-L1-/- T cells, P 〈 0.0001). (D) BALB/c mice transplanted with WT B6 or PD-L1-/- T cells. Mice were killed on d7 post-BMT and splenocytes were analyzed for LPAM-1, CCR9, and CXCR3 expression (not shown) on donor T cells. (E-F) BALB/c mice were transplanted with B6 Ly5.2 T cells plus PD-L1-/- T cells. Mice were killed on d3 post-BMT and splenocytes were analyzed for CTLA-4 and Lag-3 expression on donor T cells. Figure 1. PD-L1-/- vs. WT donor T cells lessen GVHD lethality, independent of donor Treg function. (A) Survival of BALB/c mice with WT B6 or PD-L1-/- T cells. (B) Survival of B10.BR mice with WT B6 or PD-L1-/- T cells. (C) Survival of BALB/c mice with WT B6 or PD-L1-/- T cells, or with WT B6 or PD-L1-/- CD25 depleted T cells (recipients of WT T cells vs. WT CD25-depleted T cells, P = 0.0003; recipients of PD-L1-/- T cells vs. PD-L1-/- CD25-depleted T cells, P = 0.2306; recipients of WT vs. PD-L1-/- T cells, P 〈 0.0001). (D) BALB/c mice transplanted with WT B6 or PD-L1-/- T cells. Mice were killed on d7 post-BMT and splenocytes were analyzed for LPAM-1, CCR9, and CXCR3 expression (not shown) on donor T cells. (E-F) BALB/c mice were transplanted with B6 Ly5.2 T cells plus PD-L1-/- T cells. Mice were killed on d3 post-BMT and splenocytes were analyzed for CTLA-4 and Lag-3 expression on donor T cells. Disclosures Aoyama: CHUGAI PHAMACEUTICAL CO.,LTD: Honoraria; Mochida Pharmaceutical Co.,Ltd: Honoraria; Kyowa Hakko Kirin Company,Limited: Honoraria. Milone:Novartis: Patents & Royalties, Research Funding. Miller:Coronado: Speakers Bureau; BioSciences: Speakers Bureau; Celegene: Speakers Bureau. Sharpe:Costim Pharmaceuticals: Patents & Royalties.

Abstract: Peripheral blood (PB) CD34+ cells mobilized by granulocyte colony- stimulating factor (G-CSF) administration are potentially useful for transplantation and as a target of gene transfer for therapy of hematopoietic disorders. Efficient harvest and planning for clinical use of PB CD34+ cells ideally requires foreknowledge of the expected mobilization kinetics and yield. We developed a sensitive flow cytometric assay for accurately enumerating CD34+ cells throughout the range seen at baseline to peak mobilization. We used this assay to assess the kinetics of G-CSF-mediated mobilization of CD34+ cells to PB in normal volunteers and in patients with chronic granulomatous disease (CGD) or adenosine deaminase (ADA)-deficient severe combined immunodeficiency disease (SCID). Two dose levels of G-CSF were examined (5 and 10 micrograms/kg/d for 7 days). Both doses were well tolerated. For normal subjects and patients an increase in PB CD34+ cells was first detected only preceding the third dose of G-CSF (day 3), peaked transiently on day 5 or 6, and then decreased thereafter despite additional doses of G-CSF. With 32 normal volunteers mean peak CD34+ cell counts were 57 and 76 cells/mm2 of blood (5 and 10 micrograms doses, respectively), whereas for 18 CGD patients the mean peaks were 31 and 40 cells/mm2 of blood. For 2 ADA-deficient SCID patients studied at a G-CSF dose of 5 micrograms/kg/d, the average peak was 16 cells/mm2 of blood. For both of these patient groups mobilization of CD34+ cells to PB was impaired compared with similarly treated normal subjects (P 〈 .05). By contrast to the kinetics of the CD34+ cell mobilization, the absolute neutrophil count (ANC) increased markedly by 6 hours after the first dose of G-CSF and then increased steadily through day 8. At days 5 and 6 (peak mobilization of CD34+ cells) the mean ANC of CGD and ADA patients was only slightly lower ( 〈 or = 15%) than that seen with normal subjects, whereas the difference in CD34+ cell mobilization was 〉 48%. Thus, ANC is not a reliable surrogate to predict peak PB CD34+ cell counts and direct enumeration of PB CD34+ counts should be undertaken in decisions regarding timing and duration of apheresis to harvest a specific number of these cells. Finally, unexpected, but significant differences in the PB CD34+ cell mobilization between normal subjects and patients with inherited disorders can occur and underscores the importance of establishing the expected mobilization of PB CD34+ cells in the planning of treatment approaches using these cells.

Abstract: The overall survival (OS) has improved significantly in multiple myeloma (MM) over the last decade with the use of proteasome inhibitor and immunomodulatory drug-based combinations, followed by high-dose melphalan and autologous hematopoietic stem cell transplantation (auto-HSCT) and subsequent maintenance therapies in eligible newly diagnosed patients. However, clinical trials using auto-HSCT followed by lenalidomide maintenance have shown an increased risk of second primary malignancies (SPM), including second hematological malignancies (SHM). We evaluated the impact of SPM and SHM on progression-free survival (PFS) and OS in patients with MM after auto-HSCT using CIBMTR registry data. Adult patients with MM who underwent first auto-HSCT in the United States with melphalan conditioning regimen from 2011 to 2018 and received maintenance therapy were included (n = 3948). At a median follow-up of 37 months, 175 (4%) patients developed SPM, including 112 (64%) solid, 36 (20%) myeloid, 24 (14%) SHM, not otherwise specified, and 3 (2%) lymphoid malignancies. Multivariate analysis demonstrated that SPM and SHM were associated with an inferior PFS (hazard ratio [HR] 2.62, P  & lt; .001 and HR 5.01, P  & lt; .001, respectively) and OS (HR 3.85, P  & lt; .001 and HR 8.13, P  & lt; .001, respectively). In patients who developed SPM and SHM, MM remained the most frequent primary cause of death (42% vs 30% and 53% vs 18%, respectively). We conclude the development of SPM and SHM leads to a poor survival in patients with MM and is an important survivorship challenge. Given the median survival for MM continues to improve, continued vigilance is needed to assess the risks of SPM and SHM with maintenance therapy post–auto-HSCT.

Abstract: Background: Outcomes remain dismal for patients (pts) with relapsed/refractory (R/R) AML. Programmed Death-1 (PD-1), an inhibitory receptor on T and B cells, suppresses immune activation. We hypothesized that administration of pembrolizumab, a monoclonal antibody targeting PD-1, after high dose cytarabine (HiDAC) salvage chemotherapy would stimulate a T-cell mediated anti-leukemic immune response leading to improved efficacy in R/R AML. Methods: We are conducting a multicenter phase II study of HiDAC ( 〈 60 years: 2 gm/m2 IV Q12hours days 1-5; ≥60 years: 1.5 gm/m2 IV Q12hours days 1-5) followed by pembrolizumab 200 mg IV on day 14 in R/R AML pts 18-70 years. The primary objective of this study is to estimate the overall complete remission (CR + CRi) rate. Overall responders can receive maintenance phase pembrolizumab 200 mg IV Q3weeks for up to 2 years until progression. Allogeneic stem cell transplant (SCT) is permissible before or after maintenance phase. Results: To date, 26 pts are evaluable for safety and response (Table 1). Grade ≥3 immune-related adverse events have been rare and self-limiting. The overall CR rate is 35%. Of the 9 CR pts, 5 (56%) had no evidence of minimal residual disease (MRD) by standard monitoring. Notably, 2/3 primary refractory pts with inv(3) cytogenetics achieved CR and underwent SCT. Additionally, 1 pt with relapsed AML with most recent treatment refractory to HiDAC salvage achieved CR with no evidence of MRD. Five pts received maintenance pembrolizumab: 3 relapsed (median duration of CR = 2.8 months; range: 2-5.7 months), 1 proceeded to SCT after 2 cycles, and 1 initially achieved a partial remission (PR) and had stable disease for 12 cycles of maintenance pembrolizumab before progressing. Four pts received a SCT in CR (n=3) and morphologic leukemia free state (MLFS) (n=1). Grade II acute graft-versus host disease (GVHD) and moderate chronic GVHD was seen in 2/4 (50%) pts, respectively. With a median follow up of 10.8 months to date, median overall survival is 10.5 months (range: 1.8-20.8 months). We performed B cell receptor amplicon sequencing, T cell receptor (TCR) amplicon sequencing from CD8+ T cells, and RNA-seq from enriched blasts and non-blast fractions in bone marrow (BM) from 3 CR and 3 non-responders (NR) prior to HiDAC. From the BM blast enriched fraction, expression of innate immune genes such as NLRP12, C3, S100A9, S100A12 and CD14 correlated with response. Gene Set Enrichment Analysis (GSEA) demonstrated that CR correlated with expression of genes in the Toll Pathway, Lysosome pathway, and adaptive immune system while NRs were associated with expression of the PAR1 and GATA3 pathways. From the non-blast BM population, CR correlated with expression of cell-cycle genes such as CCNE1, CCNB2, E2F2, KIF18B and CDKN3. Not surprisingly, GSEA revealed that expression of cell cycle pathways correlated with response. CR correlated with increased expression of B cell metagenes and the inverse IPRES signature in the non-blast BM fraction. As previously reported, there was a significant increase in peripheral blood (PB) TCR diversity in CR pts. CR was also significantly associated with the abundance and richness of the non-blast BM fraction of the B cell heavy and both light chains suggesting that a broader immune response at baseline may be critical for response to HiDAC and pembrolizumab. Finally, CR correlated with increased expression of CD300E, CCR7, CCR4, CCR8 and CCL7 in PB CD8+ T cells suggesting that migration of monocytes, T and B cells is associated with response. Conclusions: Our findings demonstrate that pembrolizumab is well tolerated after HiDAC in an ongoing study in R/R AML. An encouraging response rate has been seen in a high-risk patient population without apparent additive toxicity post-SCT. Our RNA-seq and amplicon sequencing data indicate that biomarkers of response are present prior to therapy. Increased expression of innate immune genes expressed by leukemic blasts and cell cycle genes by the non-blast fraction correlated with response to therapy. Additionally, CR was associated with increased measures of B and T cell diversity and immune cell migration. Further immunogenomic biomarker correlates are ongoing to determine predictors of response to pembrolizumab after HiDAC in R/R AML. Disclosures Zeidner: Tolero: Honoraria, Other: Travel Fees, Research Funding; Asystbio Laboratories: Consultancy; Merck: Research Funding; Takeda: Other: Travel fees, Research Funding; Rafael Pharmaceuticals: Other: Travel Fees; Celgene: Honoraria. Vincent:Merck: Research Funding. Foster:Celgene: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding; Shire: Honoraria. Coombs:Incyte: Other: Travel fees; AROG: Other: Travel fees; DAVA Oncology: Honoraria; Abbvie: Consultancy; H3 Biomedicine: Honoraria. Luznik:WIndMIL Therapeutics: Equity Ownership, Patents & Royalties. Gojo:Merck inc: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Serody:Merck: Research Funding.

Abstract: Thrombomodulin (TM) is a predominantly endothelial transmembrane glycoprotein that modulates hemostatic function through a domain that controls thrombin-mediated proteolysis and an N-terminal lectin-like domain that controls inflammatory processes. To test the hypothesis that TM is a determinant of malignancy and dissect the importance of these functional domains in cancer biology, metastatic potential was evaluated in TMPro mice expressing a mutant form of TM with reduced thrombin affinity and TMLeD mice lacking the N-terminal lectin-like domain. Studies of TMPro mice revealed that TM is a powerful determinant of hematogenous metastasis. TMPro mice exhibited a strongly prometastatic phenotype relative to control mice that was found to result from increased survival of tumor cells newly localized to the lung rather than any alteration in tumor growth. The impact of the TMPro mutation on metastasis was dependent on both tumor cell-associated tissue factor and thrombin procoagulant function. In contrast, expression of a mutant form of TM lacking the lectin-like domain had no significant impact on metastasis. These studies directly demonstrate for the first time that TM-mediated regulation of tumor cell-driven procoagulant function strongly influences metastatic potential and suggest that endothelial cell-associated modulators of hemostasis may represent novel therapeutic targets in limiting tumor dissemination.

Abstract: Clinical management differs significantly for the various types of non-Hodgkin lymphoma (NHL), and the diagnosis of these lymphomas can be challenging in some cases. Further, existing NHL categories include subgroups that can differ substantially in gene expression, response to therapy and overall survival. We have created a custom oligonucleotide microarray, named LymphDx, which could prove clinically useful for molecular diagnosis and outcome prediction in NHL. Biopsy specimens were obtained from 559 patients with a variety of lymphomas and lymphoproliferative conditions. Gene expression profiles of these samples were obtained using Affymetrix U133 A and B microarrays. The 2653 genes on LymphDx were chosen to include:(1)Genes most differentially expressed among NHL types based on Affymetrix U133 or Lymphochip microarrays (2)Genes predicting length of survival in diffuse large B cell lymphoma(DLBCL), follicular lymphoma(FL) and mantle cell lymphoma(MCL) (3)Genes encoded in the EBV and HHV-8 viral genomes (4)Genes encoding all known surface markers, kinases, cytokines and their receptors, as well as oncogenes, tumor suppressors, and other genes relevant to lymphoma. The LymphDx microarray was used to profile gene expression in 434 biopsy samples. These data were used to create a diagnostic algorithm that can distinguish various NHL types and benign follicular hyperplasia(FH) based on gene expression. The algorithm classifies a sample into one of the following categories: Burkitt’s lymphoma(BL), DLBCL, FL, MCL, small lymphocytic lymphoma(SLL) or FH. The algorithm further distinguishes the 3 recognized DLBCL subgroups: germinal center B cell-like, activated B cell-like or primary mediastinal lymphoma. Using a leave one out, cross validation strategy, the algorithm was found to agree well with the pathology diagnosis (see Figure). Some samples were deemed unclassified when their gene expression did not adequately match with that of any of the NHL categories. For a few samples, the gene expression-based diagnosis and the pathology diagnosis were discordant. Pathology review showed that two NHL types coexisted (eg FL and DLBCL) in many of these cases, potentially explaining the results of the diagnostic algorithm. LymphDx could also reliably predict the overall survival of patients with DLBCL, FL and MCL. Prospective evaluation of the LymphDx microarray is warranted since it could be used to provide objective molecular diagnostic, and prognostic information for patients with NHL. Figure Figure