ALBERT

Description: The evolutionarily conserved Notch receptors play important roles in cell fate decisions. Ligation of Notch-1 causes differentiation of T/NK cell precursors from HPCs and is critical for development of T cells in the thymic microenvironment. Five known Notch ligands exist in mammals (Delta1, Delta3, Delta4, Jagged1, and Jagged2), and Delta4 in particular has a greater capacity to support T cell development than Delta1. Notch ligation through Delta1 has also been shown to potentiate CD56+ NK cell differentiation from human HPCs in the presence of IL-15, although the phenotype and functionality of these cells has not been extensively described. We compared the ability of the 5 mammalian Notch ligands to induce the development of functional NK cells from human CD34+ HPCs derived from umbilical cord blood (CB). CD34+ cells isolated from CB were cultured in RPMI + 10% FBS on a murine stromal cell line, OP-9, expressing one of the five mammalian Notch receptors (Jagged1, Jagged2, Delta1, Delta3, or Delta4) or OP-9 cells transfected with vector alone, in the presence of IL-7, Flt3 ligand (FL), and IL-15. After three weeks of culture, development of CD56+CD3− cells was greatly accelerated by the ligands Jagged2 (53.4 +/− 5.5% CD56+CD3− cells), Delta-1 (38.6 +/− 5.7%), and Delta-4 (65.0 +/− 3.9%) versus culture in the absence of ligand (17.6 +/−10.3%, p = 〈 0.02) or in the presence of Jagged1 or Delta3. By 5 weeks, the percentage of NK cells seen in cultures containing Jagged2, Delta1, or Delta4 reached 80–90%. These NK cells expressed CD117 but only partially expressed CD94, with positivity ranging from 12.1 to 34.1% of NK cells derived from these 3 ligands after 5 weeks in culture; similarly, few CD16+ NK cells were seen in these cultures (0 to 12.1% of NK cells after 5 weeks). KIR expression in more than 1% of NK cells was not identified under any culture condition. In preliminary experiments, the addition of IL-2 or IL-21, both of which have been shown to induce KIR expression in non-Notch mediated models of NK cell development, did not significantly alter the percentages of NK cells expressing CD94, CD16, or KIR. Because the ligands Jagged2 and Delta4 induced the highest percentages of NK cells in culture, we examined the cytotoxic activity of these cells. NK cells derived from Jagged2 or Delta4 ligation expressed perforin and displayed in vitro cytotoxic activity against the human leukemia cell lines K562 (34.1% or 40.8% target cell lysis, respectively, at an E:T of 10:1) and HL-60 (14.1% or 31.6%, respectively). These cells also produced IFN-gamma, with Delta4 cultures producing higher levels of IFN-gamma versus Jagged2 cultures (1112 vs. 163.9 pg/ml, respectively). These data demonstrate that the Notch ligands vary in their ability to induce differentiation of NK cells from human CD34+ HPCs. Jagged2 and Delta4 in particular have greater capacity to generate functional NK cells which have cytolytic activity and can secrete IFN-gamma, while at the same time lacking a majority of inhibitory NK receptors (KIR and the NKG family of receptors which dimerize with CD94). The generation of cytolytic KIR-negative NK cells is of interest for cellular therapy against malignancies that are susceptible to NK cell killing.

Description: We hypothesized that after allogeneic hematopoietic stem cell transplant (HSCT), GvHD-affected tissues harbor expanded “immunodominant” T-cell clones, and characterization of these clones can be used to develop markers of disease. A multiplex PCR was used to detect T-cell receptor variable beta (VB) chain rearrangements in target tissue. Molecular analysis of the amplified VB CDR3 sequences allowed for identification and quantitation of putative disease-associated “clonotypes” and for the development of clone-specific PCR. We studied 5 HSCT patients for the presence of signature clonotypes in 10 skin biopsies taken during diagnostic GvHD work-up. Size distribution analysis of VB PCR products showed a skewed peak pattern in 9 biopsies; immunodominant clones (per definition frequency ≥30%) were detected in 6/7 biopsies with histologically confirmed GvHD, consistent with the presence of expanded clonotypes and the oligoclonal nature of the tissue-specific alloresponse. Immunodominant clones were also found in 2 of 3 biopsies not diagnostic for GvHD but obtained based on strong clinical suspicion, raising the possibility that they were associated with early evolving GvHD not distinguishable by histology. For example, when serial skin biopsies were analyzed, a GvHD-positive post-transplant d63 biopsy contained an immunodominant clone (frequency 60%), which was also detected in a subsequent biopsy positive for GvHD (frequency 33%). Similar results were seen in another patient, in whom serial biopsies taken on d214 (not diagnostic) and d217 (GvHD-positive) showed an identical immunodominant clone, not present in a d13 GVHD-negative biopsy. This finding suggests that the d214 biopsy might have contained early GvHD that was not detectable morphologically. In a patient who rejected an initial MUD graft (Tx 1) and then received a MUD SCT (Tx 2) from a different, unrelated donor, immunodominant clones were identified in GvHD-positive biopsies following each transplant, that were distinct for each graft. To examine whether immunodominant clonotypes derived from biopsies could be used as markers of disease, clonotypic PCR was developed for an immunodominant biopsy-derived clonotype for each transplant. The Tx 1 clonotype was detected in blood and skin following Tx 1, but not in tissue or blood taken after Tx 2. Specificity and correct size of the clonotypic PCR product were confirmed by both Genescan analysis and sequencing. Clonotypic PCR designed for an immunodominant clonotype from the Tx 2 donor detected the putative allospecific clonotype in serial samples after the second engraftment. Neither clonotype could be found in either donor, indicating that the disease-associated clones expanded to detectable levels following transplant. These results indicate that clonotypic PCR can distinguish distinct GvHD-associated clonotypes from different donors in both blood and tissue following transplant. Monitoring of the relative frequency of disease-associated clones in recipient blood indicated a significant peripheral expansion of disease-associated clones at the time of active GvHD. Our results demonstrate an efficient method for identification of disease-associated clonotypic markers, which can be used to aid diagnosis and monitoring of GvHD.

Description: Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by osteolytic bone disease and immunosuppression. Transforming growth factor-beta (TGF-β) supports MM progression through its role in stimulation of IL-6, Th17/regulatory T cell development, hematopoietic suppression, and its promotion of catabolic bone remodeling. Inhibition of the proteasome has emerged as a powerful strategy for treatment of MM. Proteasome inhibitors (PIs) have improved overall survival in patients with MM, but the majority develop drug resistance. Therefore, a novel therapeutic strategy is needed for better treatment of MM. Vactosertib (VACTOSERTIB) is an orally bioavailable small molecule inhibitor of the TGF-β type I receptor kinase (TβRI) currently in a phase I clinical trial (NCT03143985) in combination with the immunomodulatory drug, pomalidomide. In this study, we first investigate if Vactosertib is capable of diminishing myeloma progression either as a single agent or in combination with the PI, ixazomib. Methods: Expression levels of TGF-β1 and phospho-Smad2/3 was examined in human and mouse MM cells and bone marrow by Western blot and immunohistochemistry (IHC). Cellular and molecular assays were performed in human RPMI8226, ARH-77 and PIs-resistant RPMI8226 as well as murine 5T33MM cells via MTT, apoptosis, real-time PCR and Western blot. For preclinical evaluation, C57BL/KaLwRij mice bearing 5T33MM cells expressing luciferase were treated with Vactosertib, ixazomib, or the combination of Vactosertib plus ixazomib for 3 weeks, and evaluated for MM growth by bioluminescence imaging (BLI). Peripheral blood IL-6 and monoclonal protein concentration, M-spike, were measured by ELISA. Fluorescence-activated cell sorting (FACS) analyses were used for immunological evaluation, such as myeloid derived suppressor cells (MDSCs), Foxp3+ regulatory T cells (Tregs) and NK cells. Femur trabecular bone density and microarchitecture were assessed by 3D micro-CT system. Results: We first evaluated the expression of the TGF-β1 ligand and its receptor activated signaling intermediates, Smad2/3 in bone marrow of patients with MM. There is an accumulation of the type 1 isoform of the TGF-β ligand within the tumor microenvironment (TME) and of phospho-Smad2/3 in myeloma cells within the bone marrow of patients (Figure 1). In vitro studies utilizing the TbRI inhibitor Vactosertib (TEW-7197) with murine 5T33MM, human RPMI8226 (Figure 2A), U266, ARH-77 cell lines, and tumor cells from patients with MM (Figure 2B) cultured demonstrate a dose-dependent reduction in cell viability in cultures of these MM cells continuously exposed to Vactosertib. Synergistic inhibition of Vactosertib in combination with PIs was also observed in parent (Figure 2C), bortezomib- (Figure 2D), ixazomib- (Figure 2E), and carfilzomib-resistant RPMI8226 cells (Figure 2F). We also found that there is an accumulation of the TGF-β1 within the TME and phospho-Smad2/3 within the MM at advanced stages in the bone marrow of C57BL/KaLwRij mice harboring the 5T33MM myeloma cells. Oral administration of Vactosertib as a single agent inhibited MM progression as measured by mortality (Figure 3A), peripheral blood monoclonal protein concentration and BLI (Figure 3B). While the expansion of CD11b+Gr-1+ MDSCs (Fig. 3C) and the population of Tregs (Fig. 3D) in the spleen were reduced, NK cells were increased by Vactosertib alone (Fig. 3E). Combination therapy of Vactosertib plus ixazomib exhibited a synergistic anti-tumor effect when compared to either Vactosertib or ixazomib alone by a decrease in mortality (Figure 4A) and significant reduction in both the BLI and M-spike before and after treatment (Figure 4B, C). Furthermore, Vactosertib single therapy enhanced the bone thickness and reduced trabecular separation in comparison to control mice. Conclusions: Taken together, our data demonstrate that Vactosertib (a small molecule inhibitor of the TGF-β type I receptor kinase) suppresses viability of human MM cells, and is associated with a potent anti-myeloma effect, suppressing bone resorption and modulating the MM TME in a immunocompetent preclinical model of MM. These data provide a rationale for clinical evaluation of the combination therapy of Vactosertib and ixazomib as a potential therapeutic strategy to improve outcomes in patients with MM. Disclosures Kim: MedPacto: Other: Founder/CEO. Malek:Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau.

Description: Introduction: Very advanced age (≥80 years) DLBCL patients have worse prognosis. These unfavorable outcomes are largely considered to be a result of the combined effects of increased comorbidities, frailty, diminished tolerance and access to effective chemoimmunotherapy. It is not clear yet whether DLBCL patients of very advanced age have disease that is intrinsically more aggressive. Methods: We accessed the Stem Cell Transplant and Hematologic Malignancies database of University Hospitals Seidman Cancer Center for DLBCL patients diagnosed between 2000 and 2016. Information collected included demographic characteristics, baseline laboratory and disease information, as well as treatment details. Progression free survival and overall survival were calculated from time of diagnosis using Kaplan Meier methodology, comparisons were done with log rank. Cumulative incidence of relapse with death as competing risk was calculated for patients who underwent treatment, comparisons between age groups were done with the Gray test. Results: A total of 542 DLBCL patients were identified, 85 (16%) were older than 80 years. Table 1 shows the baseline demographic and disease characteristics. Older patients had a higher incidence of comorbidities, specifically cardiovascular comorbidities, prior renal insufficiency, and hyperlipidemia. Expectedly, very elderly patients had higher R-IPI, with a trend towards worse performance status that did not reach statistical difference. The proportion of patients diagnosed with non - germinal center DLBCL was not different than younger DLBCL patients, and there was no statistical difference in the incidence of double expressor or double hit lymphomas, possibly secondary to the small number of patients tested. A smaller proportion of patients ≥ 80 years received antineoplastic therapy (89% vs. 98%, p = 0.001), and use of less intense therapy was more common (table 2). Sixty one percent of patients, however, were still treated with R-CHOP (38.8%) or R-miniCHOP (22.2%). The overall response rate (ORR) for any therapy was 68.2%, lower than the ORR for younger patients (85.9%, p = 0.007). When only patients treated with RCHOP/R-miniCHOP were included, the difference in ORR was smaller, though still statistically significant (77.5% vs. 89.8%, p = 0.021). Median number of cycles was similar (5 vs. 6 cycles, respectively). Patients of all ages treated with single agent rituximab presented an ORR of 43%. After a median of 40 months follow up, estimated 4-year overall survival was 42% (95% CI 31-54%) for patients ≥ 80 years, 67% for patients aged between 60 and 79 years (95% CI 61-74%) and 78% for patients 〈 60 years (95% CI 73-84%) (Figure 1). Four - year survival for patients ≥80 years treated with R-CHOP or R-miniCHOP chemoimmunotherapy was 50% (95% CI 36-65%) vs. 75% for younger patients (95% CI 70-79%) (p

Description: Background: Follicular lymphoma (FL) is the second most common type of lymphoma in the United States. The clinical course is variable--some patients have an indolent course while others experience relatively aggressive disease, transformation to higher grade, and short survival. Despite a wide range of treatment options, no consensus exists concerning optimal therapy. Clinical predictors of outcome such as the FL International Prognostic Index (FLIPI) exist; however, biologic risk factors are needed to assist in providing accurate risk stratification. Recent data have suggested that the anti-tumor immune response may affect the clinical course and survival. In this study we sought to identify biologic indicators of prognosis in FL patients using a tissue microarray. Methods: Biopsies from 94 newly diagnosed FL patients who presented between 1985 and 2002 were reviewed for cytologic grade (CG)(Mann-Berard criteria, WHO classification) and presence of diffuse areas (DAs) prior to placement in a tissue microarray (two 1.5 mm cores/case). Cases with diffuse large B-cell components were not included. Immune response was assessed with immunostains for CD3 and CD25 as indices of T-cell infiltration and activation, respectively, and CD68 and CD163 to evaluate macrophage infiltration. Lymphoma cells were evaluated for bcl-2 expression, as well as CD10 and MUM1 expression to assess germinal center phenotype. Initial and subsequent patient management was individualized and varied considerably. Clinical data were obtained for FLIPI and overall survival (OS). Statistical analysis was performed using P ≤ 0.10 as significant for univariable and P ≤ 0.05 for multivariable analysis. Results: The patients consisted of 50 males (53.2%) and 44 females (46.8%), with a median age at diagnosis of 58 (range 24–89) years. The FLIPI distribution was 34 low risk (0–1), 26 intermediate risk (2), and 34 high risk (〉2). There were 31 deaths. The median survival was 174.1 months and the median follow-up among surviving patients was 68.8 months (range 19.5–196.3). Cox proportional hazards analysis for risk of death showed no association with CG, DAs, or expression of bcl-2, CD3 (intrafollicular), CD25, CD68, or CD163. A high FLIPI score and lack of CD10 expression were both of borderline significance for higher risk of death (HR 2.06, 95% CI 0.86–4.92; P = 0.10 and HR 2.8, 95% CI 0.81–9.65; P = 0.10, respectively). MUM1 was expressed in 23 of 87 evaluable cases and was also associated with higher risk of death (HR 2.14, 95% CI 0.97–4.71; P = 0.059). Multivariable analysis showed that only MUM1 expression was associated with a higher risk of death (HR 2.30, 95% CI 1.04–5.12; P = 0.04). Conclusions: These data demonstrate that expression of MUM1 in FL cells, consistent with a late germinal center/post germinal center phenotype, identifies a group of FL patients with poor prognosis. We found no correlation between survival and host immune response, using individual analysis of immunohistochemical markers of T-cell infiltration/activation (CD3 and CD25) or macrophage infiltration (CD68 and CD163). More detailed analysis of molecules involved in the transition of centrocyte to post-germinal center B-cells in FL is warranted.