bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Tyrosine Kinase Inhibitors On Spermatogenesis and Pituitary Gonadal Axis in Males with Chronic Myeloid Leukemia
Authors: Yassin Mohamed A. , Soliman Ashraf T , Elawa Ahmed S , El-Ayoubi Hanadi Rafii , Desanctis Vincenzo
Published: October 2019

Abstract 1688

Objective:

The introduction of several classes of targeted therapeutics for the treatment of chronic myelogenous leukemia (CML) raises the question of whether male fertility is affected and the degree of this affection, if any, among the different generations of tyrosine kinase inhibitors. When two drugs are equally effective, the drug with less toxic effect on fertility is favorable. Our aims were to evaluate semen parameters and pituitary gonadal function before and 4 months after starting tyrosine kinase inhibitors (TKI) namely, Dasatinib, Nilotinib, and Imatinib in patients with CML.

Design:

Prospective study.

Setting, Patients, Interventions:

We studied the effect of TKIs' first generation (Imatinib) and second generation ( Dasatinib and nilotinib) on semen parameters, endocrine functions in 20 euogonadal male patients with CML, and capacity to ejaculate, aged from 35 to 51 years. They were receiving either Imatinib, Dasatinib or Nilotinib as upfront therapy. We studied gonadotrophins (LH and FSH) and testosterone (T) secretion and evaluated sperm parameters before and after four months of using these TKIs.

Main Outcome Measures and Results:

Four months after starting TKIs there were significant decreases in serum testosterone, LH, FSH concentrations. The total sperm count, total and rapid progressive sperm motility, and % sperms with normal morphology decreased significantly versus before treatment. (table 1). After 4 months of therapy, Dasatinib effects on sperm count (SC), volume(SV), all sperm motilities and % of sperms with normal morphology(%NM) were significantly less harmful compared to Imatinib and Nilotinib. (Table 2).

Significant correlations were found between serum T concentrations and semen parameters before and after TKIs therapy including SC ( r = 0.658 and r = 0.73 respectively, p < 0.001), rapid progressive motility (r = 0.675 and r = 0.758 respectively p < 0.001), and the % NM(r = 0.752 and r = 0.834 respectively, p < 0.001). After TKIs therapy, LH were correlated significantly with T concentrations ( r = 0.434, p < 0.001) and SV and SC (r = 0.439 and r = 0.376 respectively, p: 0.01).

Conclusion:

Our study suggests that in patients with CML TKIs are associated with significant decrease of sperm parameters and decreased concentrations of serum T, LH, FSH. These potentially toxic effects on spermatogenesis are less prominent in patients treated with Dasatinib compared to Imatinib and Nilotinib. The mechanisms and pathways for these effects need further human and/or experimental studies.

Disclosures:

Yassin: Hamad medical corporation MRC: Employment, Research Funding. Soliman:Hamad medical corporation MRC: Employment, Research Funding. Elawa:Hamad medical corporation MRC: Employment, Research Funding.
Risk of Thromboembolic Events in ET and Early/Prefibrotic PMF: The Relevance of an Accurate Histological Diagnosis
Authors: Rupoli Serena , Goteri Gaia , Picardi Picardi , Canafoglia Lucia , Micucci Giorgia , Scortechini Anna Rita , Federici Irene , Giantomassi Federica , Lio Lidia Da , Zizzi Antonio , Leoni Pietro
Published: October 2019

Abstract 1735

Background:

Essential Thrombocytemia (ET) is a myeloproliferative neoplasm characterized by increased risk of vascular events. Established thrombosis risk factors are age and previous vascular events. The clinical and prognostic relevance of WHO histologic criteria for ET and prefibrotic/early Primary Myelofibrosis (PMF) has been well recognized. Our aim was to evaluate the correlation between histologic interpretation and vascular events in our series of thrombocytemias.

Material and methods:

From our files, we retrieved all patients consecutively diagnosed as having ET with complete clinical data (N = 283) who had undergone to a bone marrow trephine biopsy before any treatment at or within 1 year of diagnosis (N= 133). The histologic slides were reviewed in order to separate true ET cases from early/prefibrotic PMF; vaso-occlusive events at diagnosis and in the follow-up were than compared in the two groups.

Results:

Histologic review reclassified 61 cases as ET and 72 cases as prefibrotic/early PMF. Prefibrotic/early PMF showed a significant higher prevalence of thrombosis history and thrombotic events at diagnosis, and an increased leukocyte count than ET (22% vs 8%, 15.2% vs 1.6%, 8389/mmc vs 7500/mmc, respectively); furthermore, venous thromboses (mainly atypical) were relatively common in PMF, as opposed to WHO-defined ET. During follow-up, patients with prefibrotic PMF, although younger, showed a significant higher risk of developing thrombosis: the 15-year risk of thrombosis was 48% in prefibrotic PMF (grade 0), 16% in early PMF (grade 1, 2) and 17% in ET. Multivariate analysis confirmed that age and histopathology are independent risk factors for thrombosis during follow-up. Patients older than 60 or with prefibrotic PMF are high risk patients whereas those younger and with non prefibrotic PMF or ET should be considered at low risk (20-year risk of thrombosis 47% vs 4%, p=0.005).

Conclusion:

The results of present study indicate prefibrotic PMF as a myloproliferative neoplasm with the highest tendency to develop vascular events compared to early PMF and ET. Therefore we suggest to include histopathology interpretation in the risk stratification of so-called ET patients.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of Mitogen Activated Protein Kinase Kinase (MEK1) Is Effective Against CLL Cells Cultured in Media Alone or in a Supportive Microenvironment and Is Synergistic with Fludarabine in a Mechanism That Involves Decreased Levels of Reactive Oxygen Species and MCL-1 Protein
Authors: Best Giles , Crassini Kyle , Stevenson Williams , Mulligan Stephen P.
Published: October 2019

Abstract 1804

Background

Despite the high response rates of patients with Chronic Lymphocytic Leukemia (CLL) to the fludarabine (F), cyclophosphamide (C), rituximab (R) regimen, relapsed or refractory disease is common. Novel therapeutic approaches are required that are effective in this setting.

Targeting specific signaling molecules is proving an effective strategy for treating patients who are refractory to FCR. Given that the mitogen-activated protein kinase pathway (MAPK) pathway is constitutively active in CLL cells and that inhibitors of mitogen-activated protein kinase kinase (MEK1) in this pathway are in clinical trials for solid tumors, we sought to investigate the potential of MEK1 as a therapeutic target in CLL.

Results

Inhibition of MEK1/2 using MEK inhibitor I (MEKi; Calbiochem/Merck) induced apoptosis in the MEC1 cell line and in 18 patient samples. Importantly, sensitivity of the patient samples occurred irrespective of ATM/TP53 functional status, of poor prognostic features or of treatment history. MEKi was also effective against 4 CLL patient samples cultured in an in vitro model of the tumor microenvironment, albeit with a significantly higher IC50 than observed against CLL cells cultured in media alone.

As fludarabine-based therapies have become the mainstay of CLL treatment, we investigated the effect of combining the MEK inhibitor with this purine analogue. Synergy between MEKi and fludarabine was apparent against the MEC-1 cell line and 10 patient samples. Dose reduction indices (DRI) calculated from the drug combination indicate this synergy was predominantly due to an increase in fludarabine sensitivity. Investigation of the mechanisms of the synergy between MEKi and fludarabine suggests decreased levels of reactive oxygen species (ROS) and expression of the pro-survival protein, MCL-1, may be contributing factors (see figure).

These data suggest for the first time that inhibition of MEK1/2 may represent a potential therapeutic option for CLL patients. The efficacy of the MEK inhibitor against CLL cells cultured in the supportive in vitro environment suggest that this approach may also be effective at targeting the proliferative fraction of CLL cells in the tumor microenvironment. As clinical trials of MEK1/2 inhibitors are currently underway in solid tissue malignancies, our data suggest that trials of these agents may also be warranted for high risk CLL.

Disclosures:

No relevant conflicts of interest to declare.
The Chromosomal Abnormalities Del(17p), t(4;14), and +1q21 Predict Progression From Smoldering to Symptomatic Multiple Myeloma
Authors: Neben Kai , Jauch Anna , Hielscher Thomas , Hillengass Jens , Lehners Nicola , Raab Marc S , Ho Anthony D. , Goldschmidt Hartmut , Hose Dirk
Published: October 2019

Abstract 1806

Background:

Smoldering Multiple Myeloma (SMM) is a plasma cell disorder defined by the presence of ≥10% plasma cells in bone marrow and/or a monoclonal protein level of ≥3 g/dl in serum without organ damage. The aim of the study was to analyze the prognostic impact of chromosomal aberrations on time to progression (TTP) from SMM to symptomatic MM.

Design and Methods:

For selection of the patients, we used the same criteria as previously described by Kyle (Kyle et al., NEJM, 2007). We analyzed the prognostic value of 5 chromosomal abnormalities and hyper-/non-hyperdiploidy (HD and NHD, respectively) in a series of 231 patients with SMM by fluorescent in situ hybridization (FISH). Gains of at least 2 of the 3 chromosomes 5, 9, and 15 defined HD status.

Results:

Interphase-FISH analysis on CD138-enriched plasma cells detected gains of chromosomes 1q21 (29.4%) as well as deletions of chromosomes 13q14 (19.3%) and 17p13 (6.1%). Furthermore, the IgH-translocations t(4;14) and t(11;14) were observed in a frequency of 9.2% and 22.3%, respectively. The presence of t(4;14) was correlated with the serum heavy chain IgA (p<0.001). For the entire group, the median TTP was 4.9 years (95% CI, 3.9 – NA). Of all analyzed chromosomal abnormalities, del(17p13), t(4;14), and +1q21 showed a significant impact on TTP, whereas the presence of t(11;14) and del(13q14) was of no statistical significance. The median TTP for patients with del(17p13) was 2.7 years (vs. 4.9 years without, p=0.019), with t(4;14) 2.9 years (vs. 5.2 years without, p=0.021), and with +1q21 3.7 years (vs. 5.3 years without, p=0.013), respectively. In addition, HD was associated with a statistically shorter median TTP of 3.9 vs. 5.7 years in patients with NHD, respectively (p=0.036). A multivariate analysis identified t(4;14), +1q21, HD, reduction of uninvolved immunoglobulins (no.), and the risk score defined by Kyle et al. as independent factors for adverse outcome.

Conclusions:

The study shows that the overall risk of progression in SMM is significantly influenced by markers for tumor burden (i.e. Kyle risk score) as well as the presence of the chromosomal aberrations del(17p13), t(4;14), and +1q21. Our findings provide evidence that specific chromosomal aberrations are not only associated with early tumor progression and drug resistance in patients with overt MM but also to drive transition from asymptomatic into symptomatic stage of disease.

Disclosures:

No relevant conflicts of interest to declare.
Microfluidic Platform Enabling Primary Multiple Myeloma Mono- and Cis-Co-Culture Analysis
Authors: Chorom Pak , Young Edmond WK , Callander Natalie , Beebe David J , Miyamoto Shigeki
Published: October 2019

Abstract 1830

Multiple myeloma (MM) is a universally fatal disease, comprising 15% of hematological malignancies and 1% of all cancers. It has a median survival of 5–7 years from diagnosis. While newer drugs, such as the proteasome inhibitor bortezomib, have increased response to therapy, resistance and relapse still remain a key concern. Therefore, there is a need to understand the mechanism(s) of drug resistance. Ideally, this needs to be accomplished in a patient-specific manner. One approach is to directly examine biological responses of patient myeloma cells in the presence of their own stromal components under different drug conditions. However, individually collected patient bone marrow samples yield a limited number of MM tumor and associated stromal cell types, which limit the number of conditions as well as replicates that can be performed for such functional analyses.

To address this technical barrier, we have developed a first generation functional microfluidics-based microchannel platform that enables the analysis of these limited samples in mono- and/or cis-coculture (Figure 1). CD138+ patient myeloma cells are seeded into the central channel, which has a well to retain the suspension MM cells. CD138- “stromal” or mononuclear cells are seeded into each side channel. Each side channel communicates with the central channel via diffusion ports, which are only large enough for media and soluble factors to flow through. In this manner, both without (monoculture) and with the patients own stromal components (cis-co-culture) can be analyzed for MM cell responses.

Here we employed this assay platform to analyze CD138+ MM tumor cells' drug responses to the proteasome inhibitor bortezomib/Velcade and whether CD138- mononuclear cells from the same patient provide a protective effect. We added 7500 CD138+ in the central channel with or without 8000 CD138- mononuclear cells in each side channel, which were obtained from the same bone marrow aspirates. We were able to measure dose-dependent reductions in cell viability in multiple patient MM cells. Such MM cell viability was protected by the presence of patients' own CD138- mononuclear cells to varying degrees.

More patient samples are currently being analyzed and will be similarly assessed to determine whether such in vitro bortezomib responses have any relevance to patient drug responses in the clinic. Additional improvements will be incorporated with the goal to make the assay platform more robust and quantitative in its predictive capacity.

Disclosures:

Beebe: Bellbrook Labs: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
GATA1 and the BET Family Protein Brd3 Form a Mitotic Bookmarking Complex
Authors: Kadauke Stephan , Campbell Amy E , Stonestrom Aaron J , Jain Deepti P , Hardison Ross C , Blobel Gerd A
Published: October 2019

Abstract 282

Erythroid-specific transcription patterns are maintained throughout cell division. During mitosis, transcription is silenced globally. This raises the question whether mechanisms are in place that ensure the spatially and temporally correct reassembly of transcriptional regulators and thus maintain lineage fidelity. We recently found that, in contrast to most nuclear regulators, the master hematopoietic regulator GATA1 remains associated at a subset of its targets within mitotic chromosomes in erythroid cells (Kadauke et al., Cell 2012). GATA1 appears to function by creating an epigenetic “bookmark” to facilitate timely post-mitotic transcription reactivation of its mitotic target genes.

GATA1 is acetylated at two lysine-rich domains near its zinc finger domains. We recently discovered that acetylated GATA1 recruits the double bromodomain protein Brd3 to erythroid target genes (Lamonica et al., PNAS 2011). Brd3 interacts with acetylated GATA1 via its first bromodomain, and Brd3 recruitment to GATA1 target sites is critically required for induction of terminal erythroid target genes such as α- and β-globin. Notably, Brd3 belongs to a family of proteins (called the BET family) of which two members (Brd2 and Brd4) are known to be retained on mitotic chromosomes.

We now find by immunofluorescence and live cell confocal imaging that Brd3 globally binds to mitotic chromosomes. ChIP-seq experiments demonstrate a high degree of co-localization of Brd3 and GATA1 genome-wide both in interphase and in mitosis. We further demonstrate that GATA1 directly recruits Brd3 to mitotic GATA1 target sites. Transient mitosis-specific disruption of the Brd3-GATA1 interaction using the small molecule BET bromodomain inhibitor JQ1 removed Brd3, but not GATA1, from mitotic binding sites and led to a profound delay in the reactivation of GATA1-bookmarked genes. This suggests that Brd3 is an integral component of GATA1's bookmarking function. In concert, these studies support a requirement of mitotic bookmarking by a GATA1/Brd3 complex for the propagation of lineage-specific transcription programs in dividing erythroid cells.

Disclosures:

No relevant conflicts of interest to declare.
Critical Role of the Gab2/PI3K/mTOR Pathway in the Pathogenesis of Ptpn11 (Shp2) Mutation-Induced Myeloproliferative Disease.
Authors: Liu Wei , Liu Xia , Qu Cheng-Kui
Published: October 2019

Abstract 2856

Germline and somatic mutations (heterozygous) in Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling processes, have been identified in juvenile myelomonocytic leukemia (JMML), a childhood myeloproliferative disease (MPD), and pediatric acute leukemias. These mutations cause hyperactivation of Shp2 catalytic activity and enhance the binding of mutant Shp2 to signaling partners. Ptpn11 mutations are sufficient to drive the development of JMML-like MPD and acute leukemias in mice, suggesting that they play a causal role in the pathogenesis of hematological malignancies. However, the mechanisms by which Ptpn11 mutations induce these malignancies are not completely understood and the signaling partners that mediate the pathogenic effects of Ptpn11 mutations have not been explored. We previously generated a line of conditional knock-in mice with Ptpn11E76K mutation, the most common and most active Ptpn11 mutation found in JMML and acute leukemias. Induced knock-in of this mutation in hematopoietic cells resulted in MPD with full penetrance as a result of aberrant activation of hematopoietic stem cells (HSCs) and myeloid progenitors (J. Exp. Med., 2011). Recently, we discovered that the interaction between Shp2 E76K and Gab2, a prominent interacting protein of Shp2 and a scaffolding protein important for cytokine-induced PI3K/Akt/mTOR signaling, was greatly enhanced, and that mTOR was highly activated in Ptpn11E76K/+ MPD cells. To address the role of Gab2 and mTOR in the pathogenesis of Ptpn11E76K/+ mutation-induced MPD, Ptpn11E76K/+/Gab2-/- double mutant mice were generated and their phenotypes were compared with those of Ptpn11E76K/+ single mutant mice. MPD phenotypes were markedly attenuated in Ptpn11E76K/+/Gab2-/- double mutant mice. Overproduction of myeloid cells in the bone marrow was alleviated, and splenomegaly was diminished in the double mutants. Myeloid cell infiltration in the liver also decreased. Cytokine (IL-3 and GM-CSF) sensitivity of myeloid progenitors was significantly decreased in Ptpn11E76K/+/Gab2−/− mice as compared to that in Ptpn11E76K/+ mice. Hyperactivation of HSCs and excessive myeloid differentiation caused by Ptpn11E76K mutation were largely corrected by deletion of Gab2. Furthermore, we treated Ptpn11E76K/+ mice with Rapmycin, a specific and potent mTOR inhibitor, which substantially diminished MPD phenotypes. Collectively, this study reveals the essential role of the Gab2/PI3K/mTOR pathway in mediating the pathogenic effects of Ptpn11E76K/+ mutation and suggests that Gab2 and mTOR are potential therapeutic targets for the treatment of Ptpn11-associated hematological malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Human Multiple Myeloma and Breast Cancer Cells Evade Immune Rejection Through Expression of Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6.
Authors: Witzens-Harig Mathias , Hose Dirk , Jünger Simone , Pfirschke Christina , Khandelwal Nisit , Seckinger Anja , Conrad Heinke , Brackertz Bettina , Reme Thierry , Gückel Brigittte , Meissner Tobias , Hundemer Michael , Ho Anthony D , Rossi Jean-Francois , Neben Kai , Bernhard Helga , Goldschmidt Hartmut , Klein Bernard , Beckhove Philipp
Published: October 2019

Abstract 2942

Tumor-specific cytotoxic T cells are common in tumor patients, but ineffectively react against autologous tumor cells. Here, we demonstrate in multiple myeloma and breast cancer that human tumor cells escape recognition by tumor-specific CD8+ T cells through carcinoembryonic antigen-related cell adhesion molecule-6 (CEACAM-6) expression. We demonstrate for the first time CEACAM-6 expression in primary and established myeloma and examined the effects of altered CEACAM expression on cytotoxic T cell activity and cytokine secretion against myeloma and breast cancer cells in vitro —, and in vivo, using a xenotransplant mouse model. Cytotoxic T cells from multiple myeloma patients reacted against myeloma antigens presented by dendritic cells, but not against autologous myeloma cells, which expressed CEACAM6. Gene knockdown or blocking of CEACAM6 on myeloma cells restored CD8+ T-cell reactivity against malignant plasma cells. SiRNA-mediated CEACAM6 knockdown or inhibition by specific mAbs also restored cytokine secretion, cytotoxic activity, and antigen-specific lysis of CEACAM6-positive breast cancer cells. Moreover, CEACAM-6 inhibition was a prerequisite for efficient treatment of xenotransplanted breast tumors by adoptive T cell transfer. CEACAM6 thus plays an important role in inhibiting CD8+ T-cell responses against hematological and epithelial human tumors. Therapeutic targeting of CEACAM6 may be a promising strategy for improving cancer immunotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Inducible Costimulator Gene Transduced Bone Marrow Derived Mesenchymal Stem Cells Attenuate the Severity of Acute Graft-Versus-Host Disease in a Mouse Haploidentical Model.
Authors: Yang Dan , Wang Jian-Min , Wang Li-ping , Zhou Hong
Published: October 2019

Abstract 2996

It is reported that both block of costimulator (ICOS) or its ligand signal pathway and transfusion of mesenchymal stem cells (MSCs) can reduce the severity of acute graft-versus-host disease (GvHD). Using a MHC-haploidentical murine model of acute GvHD, we investigated the effects of MSC-ICOS-EGFP, bone marrow derived MSCs transduced with ICOS-gene using an adenoviral vector, on acute GvHD. Lethally irradiated CB6F1 mice were transplanted with bone marrow cells and spleen cells from C57BL/6 mice, and injected with MSC-ICOS-EGFP, MSCs, ICOS-Ig fusion protein or the diluent (control), respectively. Survival in recipients of MSC-ICOS-EGFP was significantly higher than that in GVHD group (74.29±7.39% vs 0, P=0.000, n=35), and also higher than in MSCs, ICOS-Ig, or MSCs+ICOS-Ig groups (74.29±7.39% vs 42.86±8.36% or 48.57±8.45% or 50.43±8.45%, P=0.0039 or 0.0323 or 0.0418, n=35). The lower mortality in recipients of MSC-ICOS-EGFP was related to lower incidence of acute GvHD. MSC-ICOS-EGFP can reduce the number of CD4+T cells in the early stage of GvHD due to the increased apoptosis, and it can produce CD4+CD25+Treg cells meanwhile. The effect on GvHD in recipient of MSC-ICOS-EGFP was also associated with higher serum levels of IL-4, IL-10 and lower levels of IFN-γ, IL-2, IL-12, IL-17A, as well as inducing expression of GATA-3, STAT6 transcription factors while inhibiting expression of T-bet, STAT4, ROR-rt in spleen. In vitro, the CD4+T cells of donor were sharply inhibited by MSC-ICOS-EGFP and the B7h blockade. In MLRs we can also detect lower levels of IFN-γ, IL-2and higher levels of IL-4, IL-10. These data indicate that MSC-ICOS-EGFP is a more prospective strategy for acute GvHD prevention, which had a synergism effect between MSCs and ICOS-Ig. The mechanisms of MSC-ICOS-EGFP on acute GvHD were closely associated with modulation of CD4+T cells, CD4+CD25+Tregs, and regulation of the Th1/Th2/Th17 balance in the early stage of GvHD.

Disclosures:

No relevant conflicts of interest to declare.
Role of Serum Lactate Dehydrogenase (LDH) As a Prognostic Marker for Autologous Hematopoietic Stem Cell Transplantation for Multiple Myeloma.
Authors: Patel Krina , Orlowski Robert Z. , Shah Nina , Bashir Qaiser , Parmar Simrit , Dinh Yvonne T , Rondon Gabriela , Giralt Sergio A , Champlin Richard E. , Qazilbash Muzaffar
Published: October 2019

Abstract 3115

Background:

The International Staging System (ISS), chromosomal abnormalities, and response to therapy are well recognized predictors of outcome in multiple myeloma (MM). However, the role of serum lactate dehydrogenase (LDH) as a prognostic marker for MM is not well established. Recently we showed that high LDH at diagnosis of MM is a predictor of shorter survival. Here we report the impact of the LDH level at the time of autologous hematopoietic stem cell transplantation (auto-HCT) on its outcome.

Methods:

We evaluated 1,658 patients with symptomatic myeloma who underwent auto-HCT from July 1988 to December 2010 at our institution. The primary objective was to determine the impact of high LDH (>1000 IU/L) level, obtained on the start day of the preparative regimen, on progression free survival (PFS) and overall survival (OS).

Results:

Patient characteristics according to LDH level at auto-HCT are summarized in Table 1. Patients in the 2 LDH groups (>1000 or ≤ 1000) were matched for age, gender, disease status, and response to prior therapy at the time of auto-HCT. Patients with LDH >1000 IU/L had a significantly higher beta-2 microglobulin (β2m) and bone marrow plasmacytosis at the time of auto-HCT. Median times to neutrophil (10 vs. 10 days: p=0.10) and platelet engraftment (11.3 vs.12.2 days: p=0.20) were not different in the 2 groups. Also, there was no significant difference in CR, VGPR, PR or overall response rates between the 2 groups. Median follow up was 35 months (1 to 244). Median OS in patients with LDH >1000 and ≤ 1000 were 49.2 and 68.0 months, respectively (p=0.03). Median PFS in patients with LDH >1000 and ≤ 1000 were 14.4 and 24.7 months, respectively (p=0.001). On univariate analyses, >10% plasma cells in bone marrow biopsy, relapsed disease, serum β2M ≥ 3.5 at auto-HCT, presence of any chromosomal abnormality, and < PR after auto-HCT were associated with significantly shorter PFS and OS.

Conclusions:

Having a serum LDH value of >1000 IU/L prior to auto-HCT is associated with shorter PFS and OS in patients with MM. These high risk patients may require aggressive post-transplant therapy, including consolidation, maintenance, tandem transplants or novel approaches like immunotherapy.

Disclosures:

Shah: Celgene: Membership on an entity's Board of Directors or advisory committees.
Risk Factor Analysis Of Non-Hodgkin Lymphoma-Associated Hemophagocytic Syndromes: A Multicenter Study
Authors: Sano Hirozumi , Kobayashi Ryoji , Tanaka Junji , Hashino Satoshi , Ota Shuichi , Torimoto Yoshihiro , Kakinoki Yasutaka , Yamamoto Satoshi , Kurosawa Mitsutoshi , Hatakeyama Naoki , Haseyama Yoshihito , Sakai Hajime , Sato Kazuya , Fukuhara Takashi
Published: October 2019

Hemophagocytic lymphohistiocytosis (HLH) is often associated with malignant diseases, mainly B-cell or T/NK-cell lymphoma. However, to date, few studies have examined lymphoma-associated hemophagocytic syndrome (LAHS). Our aim was to clarify the risk factors and prognostic factors of LAHS. A total of 1,181 patients with non-Hodgkin lymphoma were analyzed at 12 institutions in Hokkaido prefecture between April 2007 and December 2011 to assess the incidence, prognosis, and risk factors of LAHS. To evaluate the risk factors for developing LAHS, patient characteristics including age, gender, and histopathology were compared between patients with and without LAHS. The cumulative incidence rate of LAHS was 3.0% (35/1,181). The mortality rate of patients with LAHS was 69% (24/35), which was significantly higher than that of patients without LAHS (29%, P<0.001). The frequency of LAHS was higher in patients with T/NK-cell lymphoma than in patients with B-cell lymphoma (11.6 vs 1.8%, P<0.001). No significant differences were observed in age or gender between patients with and without LAHS. Patient characteristics including age, gender, histopathology, clinical symptoms, treatment for LAHS, EB virus serology, and laboratory data were subsequently compared between alive and dead patients to evaluate the prognostic factor of LAHS. The results obtained showed that the mortality rate was significantly higher in patients with T/NK-cell lymphoma than in patients with B-cell lymphoma (88 vs 53%, P=0.035). The frequency of liver dysfunction, including elevated total bilirubin (T.Bil) and liver enzymes [glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT)], was higher in fatal cases than in alive cases (T.Bil, median 1.35mg/dl, range 0.6-12.7mg/dl vs median 0.9mg/dl, range 0.5-6.1mg/dl, P=0.069; GOT, median 85.5IU/L, range 16-1,076IU/L vs median 40.0IU/L, range 10-651IU/L, P=0.076; GPT, median 62.5IU/L, range 11-910IU/L vs median 31.0IU/L, range 6-362IU/L, P=0.038). Moreover, the mortality rate of patients who did not respond to initial treatments including corticosteroids was higher than that of good responders (95 vs 54%, P=0.049). EB virus serology had no significant clinical impact on the prognosis of LAHS. In conclusion, patients with T/NK-cell lymphoma showed not only higher complication rates of LAHS than those of patients with B-cell lymphoma, but also higher mortality rates after developing LAHS. Further preclinical and clinical studies are required to understand the detailed pathogenesis of LAHS and improve the prognosis of patients developing LAHS.

Disclosures:

No relevant conflicts of interest to declare.
Adult T-Cell Leukemia/Lymphoma Cells Were Eliminated By T Cells With Anti-CD38 Chimeric Antigen Receptor Through CD38 Expression Induced By All-Trans Retinoic Acid
Authors: Yoshida Tetsumi , Mihara Keichiro , Kitanaka Akira , Shimoda Kazuya , Morishita Kazuhiro , Takihara Yoshihiro , Ichinohe Tatsuo
Published: October 2019

Patients with adult T-cell leukemia and lymphoma (ATLL) often succumb to death even though multi-anti-cancer drugs are used. Thus, it is essential for establishing a novel therapeutic strategy for ATLL. We have previously developed a chimeric antigen receptor against CD38 (anti-CD38-CAR) and showed powerful cytotoxicity of anti-CD38-CAR to B-cell lymphoma cells as well as to myeloma cells expressing CD38. Unfortunately, as CD38 is poorly expressed on the cell surface of ATLL cells, it is required to induce CD38 to apply our anti-CD38-CAR. Here, we investigated cytotoxicity of T cells transduced with anti-CD38-CAR against ATLL cell lines and cells obtained from ATLL patients through CD38 induction by all-trans retinoic acid (ATRA), which is clinically available for acute promyelocytic leukemia. We evaluated an effect of ATRA on cytotoxicity of T cells bearing anti-CD38-CAR against ATLL cells through flow cytometry. We firstly confirmed the expression of anti-CD38-CAR on human T cells retrovirally transduced (10-70%). Then, secondly, we prepared ATLL cell lines (MT-2, MT-4, S1T, Hut102, and Su9T: >95%, <5%, <5%, 15%, and <5% at CD38 expression, respectively). We co-incubated CD38-specific T cells with ATLL cell line cells for 3 days. MT-2 cells were entirely abrogated by T cells harboring anti-CD38-CAR. However, others were restrictedly succumbed to death after 3-day co-culture with T cells carrying anti-CD38-CAR. Next, we investigated whether ATRA could enhance CD38 expression on the cell surface of ATLL cell lines and exert a cytotoxicity of T cells with anti-CD38-CAR. Intriguingly, even 10nM of ATRA augmented CD38 expression in MT-4, S1T, and Hut102 cells (>80%), but not in Su9T cells. Co-culture experiments in the presence of ATRA showed that MT-4, S1T, and Hut102 but Su9T cells were efficiently eliminated by T cells bearing anti-CD38-CAR, leading to a positive correlation of cytotoxicity with CD38 expression level. We tested whether ATLL cells obtained from 3 patients were disrupted by T cells bearing anti-CD38-CAR. CD38 was expressed in the cells from patients at a variety of expression ratio (0-30%). Intriguingly, CD38 expression was significantly enhanced in ATLL cells from 2 of 3 individual patients with ATRA (>50%). And resultantly, T cells bearing anti-CD38-CAR exerted more powerful cytotoxicity against ATLL cells with CD38 enhanced by ATRA (cytotoxicity of T cells with anti-CD38-CAR in CD38-positive ATLL fraction: >90%). ATRA exerted enhancing effect on the cytotoxicity of T cells bearing anti-CD38-CAR against ATLL cells through the augmentation of CD38 expression. These results may provide us a rationale for novel clinical settings of T cells carrying anti-CD38-CAR on patients with ATLL using ATRA.

Disclosures:

No relevant conflicts of interest to declare.
Lymphoma Monocyte Crosstalk Via Hsp27 to Promote Immune Suppression and Chemotherapy Resistance
Authors: Zhang Zhe , Bulur Peggy A , Gustafson Michael , Gastineau Dennis A. , Dietz Allan B , Lin Yi
Published: October 2019

Background: Immune editing is a major mechanism used by tumors to promote its survival. We have reported previously the presence of a novel phenotype of immunosuppressive monocytes (CD14+HLA-DRlow/neg) in a number of cancers. Increased presence of these cells was associated with decreased treatment response and OS. We have demonstrated that certain tumor cells can convert normal CD14+HLA-DR+ monocytes to CD14+HLA-DRlow/neg phenotype in an IL-10 independent and a tumor specific way. These CD14+HLA-DRlow/neg monocytes, in turn, protect tumors from cytotoxic killing from chemotherapy. Here we report up-regulation of heat shock protein-27 (HSP27) as one mechanism mediating this effect.

Method: Monocytes from healthy donors were co-cultured with lymphoma cell lines (OCI-Ly3, Jeko-1, and Granta-519) with or without doxorubicin (DOX). Cultured cells were assessed for phenotype, viability and proliferation by flow cytometry. Lymphoma cells were isolated with anti-CD19 immunomagnetic beads and assayed by immunoblot for expressions of proteins regulating apoptosis. HSP27 levels in human plasma were measured by ELISA.

Results: DOX incubation induced apoptosis and decreased viability of all three cell lines; and co-culture with monocytes improved the lymphoma cell survival (for example, untreated Granta-519 had a 2.1±0.45 fold expansion, that was reduced to 0.39±0.12 when treated with DOX and 0.81±0.27 after co-culture with monocytes with DOX. p<0.05, n=11.) Co-culture with monocytes induced increased HSP27 expression in lymphoma cells. HSP27 levels were further increased in co-culture with monocytes and DOX, with corresponding decrease in cleaved Caspase-3 levels. As tumor cells can secrete HSP27, we found detectable levels of HSP27 in plasma of lymphoma patients. Increased HSP27 in plasma correlated with increased proportion of CD14+HLA-DRlow/neg monocytes in blood.

Conclusions: We have found that monocytes may promote lymphoma resistance to DOX killing by inducing increased HSP27. In turn, HSP27 from lymphoma patients may induce immune suppressive phenotype in monocytes. Together, this data demonstrates an active cross talk between monocytes and lymphoma resulting in multiple mechanisms of tumor resistance to chemo-immunotherapy.

Disclosures

No relevant conflicts of interest to declare.
A Novel Mechanism of GLI2 Mediated Regulation of IgM Secretion in Waldenström Macroglobulinemia
Authors: Jackson David A. , Smith Timothy D. , Amarsaikhan Nansalmaa , Vrabel Ann , Tolosa Ezequiel , Almada Luciana L. , Fernandez-Zapico Martin E. , Elsawa Sherine F.
Published: October 2019

Immunoglobulin (Ig) secretion by terminally differentiated B-cells is important in the immune response to pathogens. Its over production is a defining characteristic of many B-cell malignancies, including Waldenström Macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Thus, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for these diseases. Here, we define a novel pathway involving the oncogenic transcription factor, GLI2, modulating IgM secretion via regulation of IL-6 receptor alpha subunit (IL6Rα) expression in WM cells. We have previously identified components of the Hedgehog (HH)-GLI pathway as modulators of Ig secretion. Pharmacological (GANT61) and genetic (shRNA) inhibition of GLI proteins resulted in no significant effect on cell proliferation or viability, while there was a significant reduction in IgM secretion (60%) in BCWM.1 (p<0.0001), (55%) in MWCL-1 (p<0.0001) and (50%) in RPCI-WM1 (p<0.0001) cells. Interestingly, this occurred independent of HH signaling as manipulation of HH had no effect on IgM secretion, cell growth or viability. Screening for a mechanism identified IL6Rα as a downstream direct target of GLI activity. GANT61 treatment significantly reduces IL6Rα promoter activity in BCWM.1 (p=0.0002), MWCL-1 (P=0.0003) and RPCI-WM1 (p<0.0001) cells. GLI2 knockdown using two independent shGLI2 constructs resulted in reduced IL6Rα mRNA expression (p<0.0001) determined by qPCR, and IL6Rα protein expression examined by FACS analysis. Similarly, GLI2 knockdown diminished IL6Rα promoter activity in BCWM.1 (p=0.0218), MWCL-1 (p=0.0008) and RPCI-WM1 (p=0.0002) cells. Using bioinformatics analysis, we identified three candidate GLI2 binding sites in the IL6Rα promoter region. Using chromatin immunoprecipitation assay (ChIP) followed by qPCR, we confirmed that GLI2 directly binds to the IL6Rα promoter region (-1294 / -1065). Finally, we were able to rescue the reduction in IgM secretion in GLI2 knockdown group by overexpressing IL6Rα (p=0.0142). Taken together, these data reveal a novel mechanism where GLI2 modulates IgM secretion in WM through the regulation of IL6Rα expression. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.

Disclosures

No relevant conflicts of interest to declare.
PD-1 Is Expressed on B-Cells in Waldenstrom Macroglobulinemia and Promotes Malignant Cell Viability and Proliferation
Authors: Ansell Stephen M.
Published: October 2019

The tumor microenvironment plays an important role in regulating malignant cell growth and mechanisms to enhance anti-tumor immune function have been shown to improve patient outcome. Interactions between programmed death 1 (PD-1) and its ligands (PD-L1 and PD-L2) have been shown to be an important checkpoint in immune regulation. While it is well known that PD-1 is expressed on normal T cells and signaling through PD-1 inhibits T cell function, PD-1 is also expressed on a subset of B-cells but little is known about PD-1 signaling in B-cells. The goal of this study was to determine if the PD-1 is expressed on malignant B cells in Waldenstrom macroglobulinemia (WM) and whether this pathway plays a role in the survival and growth of malignant B cells in this B cell lymphoma.

Using flow cytometry, we found that the cell lines MWCL-1, BCWM.1 and RPCI, all derived from patients with Waldenstrom macroglobulinemia, expressed PD-1 to varying degrees on their cell surface. PD-1 expression in the cell lines was further confirmed by RT PCR analysis. Using flow cytometry and immunohistochemistry to examine bone marrow specimens from WM patients, we further confirmed PD-1 expression on CD19+ CD138+ malignant B-cells. Furthermore, intense staining for the ligands PD-L1 and PD-L2 was found by in bone marrows of WM patients when compared to normal bone marrow specimens. When WM cell lines are co-cultured with stromal cells engineered to express PD-L1 or PD-L2, there was a consistent increase in cell viability compared to controls. When malignant B cells from WM patients were co-cultured with stromal cells expressing the ligands, viability was unchanged but there was an increase in cell proliferation, most noticeably when cocultured with cells expressing PD-L2. To determine potential mechanisms that account for upregulation of PD-1 on malignant B-cells, we tested whether cytokines that promote WM cell growth and survival, including IL-6, IL-21 and BAFF, increased PD-1 expression. We found that WM cell lines and patient derived CD19+CD138+WM B-cells (n=4) treated with IL-21 demonstrated an increase in PD-1 expression compared to untreated controls.

We conclude that PD-1 is expressed on malignant B-cells in WM and that signaling through PD-1 may promote WM cell growth and survival. Blocking PD-1/PD ligand interactions may therefore be a potential therapeutic strategy in patients with Waldenstrom macroglobulinemia.

Disclosures

No relevant conflicts of interest to declare.
STAT3 Activation Is Preceded By Two Independent Pathways (BCR-BTK and IL6-JAK) in Mantle Cell Lymphoma
Authors: Park Hyebin , Zhao Donglu , Tsourdinis George , Ou Zhishuo , Tamayo Archito T. , Ford Richard J. , Fang Bingliang , Pham Lan V , Zhang Liang , Wang Michael
Published: October 2019

Introduction Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor outcome and therapeutic challenge. Oral single-agent ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, elicited a response rate of 68% in phase II clinical trial and has been approved by FDA for the treatment of MCL patients who received at least one prior therapy. To increase the possibility of therapeutic potential, the investigation of BTK-dependent/independent signaling pathways is needed.

Methods and Results Using both established MCL cell lines and primary MCL cells as a model system, our data demonstrated that ibrutinib effectively inhibited BTK phosphorylation along with quick STAT3 inactivation within 30 minutes of ibrutinib incubation at a dose lower than 100nM. Since the results clearly indicated that ibrutinib inhibited both BTK and STAT3 activation in MCL cells, we next elucidated the function and action between BTK and STAT3. Using a validated BTK-specific siRNA, we knocked down BTK to test the legitimacy of the relationship between BTK and STAT3. The results showed that transient knockdown of BTK significantly inhibited STAT3 phosphorylation in MCL cells. Next, the results from both immunoprecipitation and confocal microscopy showed that STAT3 was BTK-associated transcription protein indicating that BTK could function as a kinase upstream of STAT3, in a similar manner to the JAK/STAT pathway. Since STAT3 is predominately known as the downstream protein of the IL-6/JAK pathway, we examined whether there is a cross-talk between the BTK-STAT3 and the JAK-STAT3 pathways in MCL cells. After stimulation of MCL cells with IgM or IL-6, ibrutinib only inhibited IgM-induced STAT3 activation but not IL-6-induced STAT3 activation. Even with an increased dose of ibrutinib, the basal level of STAT3 phosphorylation remains detectable in MCL cells due to IL-6 autocrine. However, ibrutinib plus JAK inhibitor completely inactivated STAT3 and synergistically inhibited the growth of MCL cells. These data indicate that there are two independent pathways, BCR-BTK and IL6-JAK, which lead to STAT3 activation without cross-talk in MCL cells.

Conclusions Our results may lead to the development of more effective combination therapy to block both BCR-BTK and IL-6-JAK signaling pathways for relapsed or refractory MCL.

Disclosures

Wang: Pharmacyclics: Honoraria, Research Funding.
Comparison of Real-Time PCR and Droplet Digital PCR for the Determination of JAK2V617F Mutation in Ph'-Negative Myeloproliferative Neoplasms
Authors: Fontanelli Giulia , Baratè Claudia , Ciabatti Elena , Guerrini Francesca , Morganti Riccardo , Arici Roberta , Barsotti Sara , Petrini Iacopo , Del Re Marzia , Petrini Mario , Galimberti Sara
Published: October 2019

Myeloproliferative neoplasms (MPNs) are a group of clonal diseases associated with JAK2 mutation (V617F) in a percentage varying from 50% (Essential Thrombocytemia ET, and Primary Myelofibrosis PMF) to 95% (Polycitemia Vera, PV). The standard method for determining the V617F mutation is today represented by the Real Time PCR (RT-PCR). However, in recent years some studies have focused attention on a new method, the Droplet Digital PCR (DD-PCR), that allows an absolute quantitation of the mutated allele without any reference curve.

The main objective of this study was the comparison of two PCR methods (RT-PCR and DD-PCR) for the analysis of the JAK2V617F mutation in order to assess the sensitivity, specificity and feasibility of the two techniques.

In the study 225 patients with MPNs JAK2V617F-positive were enrolled; in 99 cases, DNA was still available for further assays and so DD-PCR tests were performed and compared with the results coming from RT-PCR (MutaQuant kit, Ipsogen, Luminy Biotech, Marseille, France).

The results show a good sensitivity for both methods; nevertheless, after appropriate dilution tests, the DD-PCR was able to detect the JAK2V617F mutation in the 5x10-4 dilution versus 5x10-3 for the RT-PCR, in front of a fully comparable specificity. A linear regression analysis showed an absolute correlation between the 2 methods (R2=0.91), and the median mutated allele burden was not different when measured by the DD-PCR or the RT-PCR (p=0.67), with the highest median allele burden observed in secondary myelofibrosis (86% by RT-PCR and 91% by DD-PCR) and the lowest one in ET (23% by RT-PCR and 25% by DD-PCR) (see Figure 1 and Figure 2), as expected.

Figure 1 Figure 1.

Figure 2 Figure 2.

Our study demonstrates that a new molecular technique, the DD-PCR, may represent a new frontier in the world of molecular techniques for the detection of JAK2 mutation with a high sensitivity and specificity. In addition the method allows obtain two types of information, qualitative and quantitative, with one analysis, saving time and at a lower cost than the standard method.

Disclosures

No relevant conflicts of interest to declare.
Heterozygosity for JAK2V617F Is a Risk Factor of Developing Post-Polycythemia Vera Myeloofibrosis?
Authors: Cacciola Rossella , Torre Maria , Cacciola Emma
Published: October 2019

Animal model studies report that heterozygous JAK2 V617F polycythemia vera (PV) has a low risk of progression to myelofibrosis. This clinical characteristic in humans is controversial. Therefore, we evaluated JAK2 V617F allele burden and myelofibrotic indicators such as reticulin level, bone marrow (BM) cellularity, CD34 levels and spleen size in 75 patients with heterozygous JAK2 V617F PV (45 males, 30 females, mean age 68 years) diagnosed according WHO criteria. Their mean duration of disease was 11 years (range, 1-26 years). All patients were on aspirin. The percentage of granulocyte mutant alleles was evaluated using a quantitative real-time polymerase chain reaction-based allelic discrimination assay. Reticulin level and BM cellularity were evaluated by ad hoc panel of 4 hematologists and 2 pathologists. CD34 levels were measured by flow cytometric assay. The spleen size was on ultrasound scan by measuring the longitudinal diameter (centimeters). Of 75 patients, 42 had allele burden > 10% (34±5%) (group 1) and 33 had allele burden < 10% (6±1%) (group 2). All patients had minor reticulin fibrosis (0.4±0.8), normal BM cellularity (76±8%), normal CD34 level (0.08±0.09%) whereas 24/42 (57%) of the group 1 and 3/33 (9%) of the group 2 had palpable splenomegaly (14±2.6 cm and 18 cm, respectively). These results suggest that heterozygosity > 10% might indicate a higher risk into the evolution of the so-called spent phase of the PV.

Disclosures

No relevant conflicts of interest to declare.