ALBERT

Description: Plasmacytoid dendritic cells (pDCs) contribute to innate antiviral immune responses by producing type I interferons (IFNs) upon exposure to enveloped viruses. However, their role in adaptive immune responses, such as the initiation of antiviral T-cell responses, is not known. In this study, we examined interactions between blood pDCs and influenza virus with special attention to the capacity of pDCs to activate influenza-specific T cells. pDCs were compared with CD11c+ DCs, the most potent antigen-presenting cells (APCs), for their capacity to activate T-cell responses. We found that like CD11c+ DCs, pDCs mature following exposure to influenza virus, express CCR7, and produce proinflammatory chemokines, but differ in that they produce type I IFN and are resistant to the cytopathic effect of the infection. After influenza virus exposure, both DC types exhibited an equivalent efficiency to expand anti–influenza virus cytotoxic T lymphocytes (CTLs) and T helper 1 (TH1) CD4+ T cells. Our results pinpoint a new role of pDCs in the induction of antiviral T-cell responses and suggest that these DCs play a prominent role in the adaptive immune response against viruses.

Description: CD4+CD25+ regulatory T cells (Tregs) suppress immune responses to alloantigens. The in vivo circulation and tissue localization of Tregs during an adaptive immune response remain unclear. We noninvasively tracked luciferase-expressing Tregs over time in an allogeneic bone marrow transplant model and demonstrated colocalization with effector T cells and initial expansion in secondary lymphoid organs before migration into inflamed tissues. Inflammation induced by irradiation and the allogeneic setting provided crucial stimuli for early Treg expansion and migration, leading to parallel reduction of effector T-cell proliferation in lymphoid organs and peripheral tissues. Treg transplants conferred long-term protection from systemic inflammatory challenge consistent with Treg in vivo survival. Suppression occurred during multiple phases of inflammation, but is optimal in the initial phase, providing protection from graft-versus-host disease while maintaining the graft-versus-tumor effect even at physiologic doses of Tregs due to their in vivo expansion, hence overcoming a major barrier to potential clinical applications of Tregs given their rarity.

Description: Key Points The Gardos channel is a potassium channel involved in red cell volume modification. A mutation in KCNN4 encoding the Gardos channel is presented as the genetic basis for a new type of hereditary xerocytosis.

Description: Interleukin (IL) 4 is a potent immunomodulatory cytokine secreted by T-helper 2 (Th2) cells and Th2 mast cells that promotes the commitment of cells. However, unregulated production and release of IL-4 can exacerbate allergic reactions and increase susceptibility to infectious organisms and viruses. Here, we present evidence that AG-490, a Janus tyrosine kinase (JAK) 2-JAK3 inhibitor, effectively blocked IL-4 gene expression and secretion in the Th2 cell line D10 that was not occurring after anti-CD3 antibody stimulation, whereas AG-490 had no inhibitory effect on production of other Th2 cytokines or cytokines synthesized by the corresponding Th1 cell line clone 29. AG-490 potently inhibited IL-4–mediated proliferation of both D10 and the IL-4–dependent cell line CT.4S. Moreover, AG-490 markedly inhibited IL-4 activation of JAK3 and blocked the downstream activation of signal transducer and activator of transcription 6, as judged by tyrosine phosphorylation, DNA binding, and transcription assays. In contrast, AG-490 did not affect tumor necrosis factor  activation of NF-κB at similar concentrations of drug. These data suggest that tyrosine kinase inhibitors that inhibit JAK3 may have previously unrecognized and selective clinical potential as immunotherapeutic drugs to treat Th2-mediated diseases driven by IL-4.

Description: INTRODUCTION: Multiple myeloma is a heterogeneous disease featured by recurrent translocations involving the IgH region. Such cytogenetic events have a driver role in early transformation of a normal plasma cell into a MM cell. Although several studies have reported the presence of limited number of other structural chromosomal events using different approaches, including conventional cytogenetics, high-resolution genome mapping, interphase fluorescence in situ hybridization (FISH) and whole exome sequencing, the full catalogue of genomic rearrangements in MM samples has never been carried out systematically. Here, we have utilized whole-genome sequencing technologies to perform a systematic, genome-wide analysis to uncover the frequency and nature of rearrangements in MM. MATERIAL AND METHODS: We performed Whole genome sequencing (WGS) using the Illumina X10 platform in 68 serial samples from 30 patients including 11 patients with smoldering myeloma, 13 newly-diagnosed patients and 44 relapsed patient samples to provide further insight into evolution of rearrangements in MM. Structural variations (translocations, deletions, inversions, internal tandem duplications, fusions) and copy number changes were analyzed using the analysis pipeline at the Wellcome Trust Sanger Institute as recently described (Nik-Zainal Nature 2016). RESULTS: We observed a total of 1295 rearrangements for a median of 27 per sample (range 2-138) including a median of 6 (range 1-36) inversions, 5 (range 1-33) internal tandem duplications, 10 (range 1-40) deletions, 7 (range 1-32) translocations and 5 fusions (0-20). While the vast majority of events was non-recurrent, the high prevalence of rearrangements at smoldering stage and in myeloma at diagnosis and further increase at the time of relapse suggest a much more complex genomic landscape than previously thought. Translocations involving the IGH locus were identified including t(11;14) in 6 (20%), t(4;14) in 4 (13%) and t(8;14) in 3 (10%) of 30 unique patients. We also report frequent involvement by light chain loci in the rearrangements. The MYC locus was recurrently affected by non-IGH rearrangements in 11/30 (36%) patients. The other main MYC partners were IGL (4/30) and IGK (2/30), while about one-third of cases were involved by rearrangements not involving immunoglobulins or other obvious partners. MYC is therefore frequently involved by rearrangements through immunoglobulin-independent mechanisms. Interestingly, many regions affected by recurrent copy number abnormalities (CNAs) were associated with rearrangements. In particular 7/14 (50%) 1q gains and 6/8 (75%) 1p deletions were involved by translocations and inversions respectively (i.e Figure 1a). Overall 15/22 chromosome 1 CNAs were associated with a specific rearrangements. A similar association between copy number changes and rearrangement breakpoints was observed among other recurrent genomic aberrations such as 6q deletions (6/12, 50%), 8p deletions (4/7, 57%) and 16q deletions (7/13, 53%). In addition to deletions, inversions, internal tandem duplications (ITDs) and translocations, we observed at least one and often more regions of chromothripsis in 10/30 (33%) patients. Chromothripsis represents a complex event characterized by localized chromosome shattering and repair occurring in a one-off catastrophic event (Korbel J. et al. Cell 2013) (Figure 1b) and known to be associated with worse prognosis in MM. In our series, chromothriptic events were always conserved during every investigated evolution process: suggesting an early onset of this complex event in myelomagenesis. CONCLUSION: We report for the first time a comprehensive catalogue of rearrangements in MM based on whole-genome sequencing data. Our data provide evidence that the genomic landscape of rearrangements in MM is very complex and heterogeneous than speculated before and besides IgH involves number of other recurrent chromosomal alteration mechanisms. These diverse aberrations, in many cases acquired early, may deregulate oncogenes as illustrated by the MYC locus. Figure 1. Figure 1. Disclosures Moreau: Celgene: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Avet-Loiseau:sanofi: Consultancy; celgene: Consultancy; amgen: Consultancy; janssen: Consultancy.

Description: Indolent systemic mastocytosis (ISM) patients have a normal life expectancy, except in the 5% to 10% of cases that progress to more advanced SM (advSM), which has a significantly poorer outcome. Mutations in genes other than KIT frequently found in myeloid neoplasms have been associated with a poorer outcome among advSM, whereas limited information exists about their frequency and prognostic impact in ISM. We investigated the frequency and prognostic impact of variants in 18 genes, found to be altered in advSM, in 322 ISM patients (median follow-up, 5.7 years) divided into discovery (n = 200) and validation (n = 122) cohorts. Overall, 71 genetic variants were detected in 55 of 322 (17%) patients. Mutated ISM cases, particularly those carrying ASXL1, RUNX1, and/or DNMT3A (A/R/D) pathogenic variant allele frequencies (VAFs) ≥ 30%, exhibited significantly shortened (P 〈 .001) progression-free survival (PFS) and overall survival (OS). Multivariate analysis showed that serum β2-microglobulin (sβ2M) levels 〉 2.5 µg/mL (hazard ratio [HR], 9.8; P = .001), together with a KIT D816V VAF ≥ 1% in bone marrow (BM) (HR, 10.1; P = .02) and pathogenic variants of A/R/D VAFs ≥ 30% (HR, 4.2; P = .02), were the best combination of independent predictors for PFS. In turn, A/R/D gene pathogenic VAF ≥ 30% was the only independent predictor for OS (HR, 51.8; P 〈 .001). Based on these variables, 2 scoring systems were constructed for risk stratification of ISM at diagnosis with significantly different 10-year PFS (100%, 91%, 0% for scores of 0, 1, ≥2, respectively) and OS (100% and 50% for scores of 0 and 1) rates.

Description: Background and Aim The use of G-banded karyotype and FISH have been standard diagnostic tools in monitoring response to treatment and disease progression in hematological disorders, including multiple myeloma. However, with the availability of array and NGS technologies in most clinical diagnostic laboratory settings, it is time to consider evaluating the use of more modern methods in diagnosing plasma cell dyscrasias. To this end, we have consented over 20 patients, most of which have evidence of disease progression to a preliminary study comparing data from RNA-Seq, SNP-CN arrays and a targeted deep sequencing cancer panel to conventional FISH and cytogenetics. Materials and Methods Patients with evidence of disease were asked to participate in an IRB approved study with full genomics consent. CD138+/- cells were isolated from bone marrow specimens and used for RNA and DNA extraction. RNA-Seq library preparation was performed using Illumina TruSeq protocols and sequenced at 50 million reads per sample using an Illumina HiSeq2000 instrument. Matching DNA samples were processed using Illumina Omni1-Quad or Affymetrix CytoScan HD SNP copy number (SNP-CN) arrays and either the Ion Torrent AmpliSeq Cancer or the Illumina TruSeq Cancer panels at a minimum read depth of 1000x. RNA-Seq data was processes using TopHat alignment and standard tools for identifying differential gene expression (Cuffdiff), mutations (ANNOVAR) and gene fusions. SNP-CN data was analyzed using the GenomeStudio, ChAS and BioDiscovery Nexus software. Ion Torrent and MiSeq data was analyzed with on-board and third party (CLC-Bio) software. All genomic data was entered into NextBio-Clinical software with relevant clinical history. Results We have completed analysis of 2 patient samples and full results are pending for more than 20 additional samples. Although our results are preliminary, we will present compelling evidence that the combination of RNA-Seq, SNP-CN array and a targeted deep sequencing cancer panels provide greater detail into molecular markers of clonal waves and potential mechanisms that drive disease progression in multiple myeloma than can be achieved with standard karyotype and FISH. These data include identification of a low-level KRAS p.G13D mutation in the background of a NRAS p.Q61K mutation that was present in both the RNA-Seq and the targeted deep sequencing data. This patient had a partial response to targeted therapy and we are in the process of evaluating if mutations that we found may have been associated response. In addition to these data, we have evidence to support that these technologies can be implemented within a standard clinical diagnostic timeline of 2 weeks or less with available infrastructure present at many academic institutions. Furthermore, we outline a plan for HIPAA-compliant longitudinal tracking of data, data sharing and data storage using commercial vendors such as NextBio and public sources such as dbVAR and dbGAP. Conclusion In order to continue to improve outcomes in patients with multiple myeloma, we need to improve our understanding of disease progression and response to treatment. This is difficult with low complexity and low resolution technologies such as karyotype and FISH. Moreover, the ability to analyze and share clinical trials data, even low complexity data, is hampered by inefficient reporting infrastructures. The implementation of genomics workflows in clinical laboratories presents many challenges, but with those challenges also comes the opportunity to provide more informative and more actionable information that can ultimately improve the quality of care. Disclosures: Rossi: Pfizer: Consultancy; Onyx: Consultancy. Kaufman:Onyx: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy; Jansenn: Consultancy; Merck: Research Funding. Boise:Onyx Pharmaceuticals: Consultancy. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy.

Description: Interleukin (IL) 4 is a potent immunomodulatory cytokine secreted by T-helper 2 (Th2) cells and Th2 mast cells that promotes the commitment of cells. However, unregulated production and release of IL-4 can exacerbate allergic reactions and increase susceptibility to infectious organisms and viruses. Here, we present evidence that AG-490, a Janus tyrosine kinase (JAK) 2-JAK3 inhibitor, effectively blocked IL-4 gene expression and secretion in the Th2 cell line D10 that was not occurring after anti-CD3 antibody stimulation, whereas AG-490 had no inhibitory effect on production of other Th2 cytokines or cytokines synthesized by the corresponding Th1 cell line clone 29. AG-490 potently inhibited IL-4–mediated proliferation of both D10 and the IL-4–dependent cell line CT.4S. Moreover, AG-490 markedly inhibited IL-4 activation of JAK3 and blocked the downstream activation of signal transducer and activator of transcription 6, as judged by tyrosine phosphorylation, DNA binding, and transcription assays. In contrast, AG-490 did not affect tumor necrosis factor  activation of NF-κB at similar concentrations of drug. These data suggest that tyrosine kinase inhibitors that inhibit JAK3 may have previously unrecognized and selective clinical potential as immunotherapeutic drugs to treat Th2-mediated diseases driven by IL-4.

Description: Background Mantle cell lymphoma (MCL) is incurable with current standard therapies, and there is no consensus for the optimal induction regimen. Bortezomib, a 26S proteasome inhibitor, is active as a single agent in this disease, and preclinical data suggest that combination with chemotherapy may be synergistic. A recent randomized trial found that maintenance rituximab after R-CHOP led to a survival benefit, suggesting maintenance strategies are worth investigating in MCL. The SWOG cancer research cooperative group conducted a phase II study (S0601) to evaluate the safety and efficacy of combining bortezomib with R-CHOP for induction, followed by bortezomib maintenance for 2 years. Methods Patients were eligible if they had previously untreated stage III, IV, or bulky stage II evaluable mantle cell lymphoma. Induction therapy consisted of six cycles of R-CHOP (375 mg/m2 rituximab, 750 mg/m2 cyclophosphamide, 50 mg/m2 doxorubicin, 1.4 mg/m2 vincristine, and 100 mg prednisone daily for 5 days) plus bortezomib 1.3 mg/m2 on days 1 and 4 of every 21 day cycle. Patients achieving at least stable disease after induction were eligible for bortezomib maintenance therapy 1.3 mg/m2 days 1, 4, 8, and 11 every 3 months for 8 cycles (one cycle was defined as 3 months for the maintenance phase). The study was designed to estimate the 2 year PFS rate to within 13% (95% CI). Results Of the 68 enrolled patients, 65 were eligible and evaluable for toxicities. The patient population had a median age of 61 (range 36-85), with 80% male sex, 15% bulky disease, 98% stage III-IV, 37% elevated LDH, and MIPI scores of: 45% low-risk, 43% intermediate risk, and 12% high-risk. In general, the treatment was well-tolerated, with 48% of patients experiencing grade 4 hematologic toxicities during induction therapy, and 38.5% grade 3 non-hematologic and 6% grade 4 non-hematologic toxicities. During maintenance therapy, 13% of patients experienced grade 3 non-hematologic toxicities. Grade 3 peripheral neuropathy was experienced by 8% of patients during induction and 2% of patients during maintenance bortezomib, and there was no grade 4 neuropathy. One patient died of toxicities possibly related to therapy: complete heart block in the setting of pneumonia and acute respiratory distress syndrome. With a median follow-up time among surviving patients of 5.9 years, 52 patients have progressed or died. The estimate of 2-year progression-free survival (PFS) was 62%, and 2-year overall survival (OS) was 85%. At 5 years, the PFS was 28% and OS was 66%. Based on prior studies, the historical 2 year PFS rate for R-CHOP alone in this population is 30%. MIPI scores were significantly associated with outcome, with 2-year PFS of 72%, 61%, and 25% for low, intermediate, and high risk MIPI groups, respectively. However, in a regression analysis to identify prognostic factors associated with a ≥ 5 year PFS, the only significant factor found was absence of splenic involvement. Additional correlative studies are planned to identify predictive biological markers associated with long-term remissions. Conclusions Combination R-CHOP with bortezomib followed by maintenance bortezomib appears to improve outcomes compared with historical data of R-CHOP alone, doubling the historical 2 year PFS rate, with nearly one third of patients achieving a PFS of 5 years or longer. These results suggest that the addition of bortezomib to induction chemotherapy and/or maintenance is promising and warrants further exploration. Support: CA32102, CA38926, and in part by Millennium Pharmaceuticals, Inc. Disclosures Off Label Use: The use of bortezomib in the induction and maintenance setting for mantle cell lymphoma will be discussed.. Fisher:Johnson & Johnson: Consultancy; MorphoSys AG: Consultancy.

Description: CD4+CD25+ Regulatory T cells (Treg) reduce the incidence and severity of acute graft-versus-host disease (GvHD) in murine models of allogeneic bone marrow transplantation (BMT) when given at the time of transplantation at a 1:1 ratio with donor effector T cells (Tcon). In our previous study with Treg trafficking, bioluminescene imaging (BLI) indicated a persistence of signal consistent with a prolonged survival of Treg in vivo following allogeneic BMT. In the current study, we evaluated the duration of Treg suppression and the impact of Treg on evolving and established GvHD. Lethally irradiated Balb/c (H2d) hosts received 5x106 T-cell depleted bone marrow (TCD-BM) cells from wild-type FVB mice (H2q) on day 0. On day 2, 3x106 splenocytes, or 1x106 purified CD4+/CD8+ T cells (Tcon) from luciferase+ transgenic mice (FVB) were infused to induce GvHD. Treg, purified from wt-FVB mice, in a 1:1 dose ratio with Tcon, were infused either on day 0, 2, 9, or 23 post-transplantation. Bioluminescence imaging was used to localize and quantitate the proliferation of Tcon in the absence or presence of Treg. Signal intensity, measured by photons/second/mouse, was significantly decreased in animals which received Treg at day 0, 2, or 9 (p-value 〈 0.05). More importantly, the greatest reduction in signal intensity occurred when Treg were given prior to the induction of GvHD by Tcon. This reduction was associated with a significantly lower clinical score for GvHD. Studies in which Treg are given up to 10 days prior to the addition of Tcon show similar findings. At day 23, when clinical GvHD was fully established in mice which received Tcon, the addition of Treg did not alter the increasing BLI signal level or the clinical course such that all animals died of GvHD (p-value=0.38). Lymphoid reconstitution was not affected by the addition of Treg prior to the induction of GvHD. In dose titration studies whereby Treg are given two days prior to the induction of GvHD, a 10-fold dose reduction in Treg was sufficient to significantly reduce Tcon proliferation and suppress clinical GvHD. We next assessed the duration of Treg suppressive effect by inducing GvHD on day 7, 14, 19 with luc+ Tcon following the infusion of Treg on day 0 of allogeneic BMT. Treg provided protection from the Tcon challenge at all 3 time points, leading to improved survival (p-value 〈 0.05). We conclude that Treg provide prolonged protection due to their ability to proliferate in vivo in an allogeneic setting. The capacity of Treg to proliferate in vivo permits a significant reduction in the number of Treg needed for adoptive transfer to induce a clinical response, a practical finding given the rarity of Treg. In addition, we conclude that Treg suppress the early proliferation of Tcon, allowing them to prevent and control evolving but not established GvHD.