bluuebunny/crossref_metadata_2025 huggingface dataset:
Single Agent Gemcitabine As a Salvage Regimen in Patients with Relapsed/Refractory Hodgkin's Lymphoma after Autologous Stem Cell Transplantation
Authors: Ozdemir Evren , Aslan Alma , Turker Alev , Barista Ibrahim , Kars Ayse
Published: November 2019

The aim of this study was to evaluate the efficacy and safety of gemcitabine as a salvage regimen in patients with relapsed or refractory Hodgkin's lymphoma after autologous stem cell transplantation (ASCT). Fifteen patients were enrolled. All patients had received ≥ 2 prior chemotherapy regimens, had an ECOG performance status ≤ 2, had adequate organ function and had undergone ASCT. Patients received intravenous gemcitabine (1000 mg/m2) on days 1 and 15, every 4 weeks. The median age was 26 years (range, 18-44) and 8 (53%) were female. Eight (53%) had primary refractory disease. All had previous platinum-based salvage chemotherapy (IIVP, 7; ICE, 5; DHAP, 3). All (100%) had relapsed/refractory disease following ASCT as their last treatment. Eleven (73%) had refractory/progressive disease after ASCT. None had previous brentuximab vedotin treatment. Median number of previous lines of chemotherapy was 2 (range, 2-3). Median number of gemcitabine cycles administered to the patients was 4 (range, 2-6). Treatment response was assessed with PET-CT in majority of the patients before and 2-3 cycles after gemcitabine, and those patients who demonstrated a response continued to receive a maximum of 6 courses of gemcitabine or bridged to allogeneic SCT. Of 15 patients, 1 (7%) had complete response, 5 (33%) had partial response and the remaining patients had refractory/progressive disease with an overall response rate of 40%. The median time to progression for responding patients was 3 months (range, 2-7 months). Two patients were successfully bridged to allogeneic SCT. Main toxicity was hematological. Grade ≥ 3 hematologic toxicity occurred in 8 patients: thrombocytopenia (40%), neutropenia (33%) and anemia (7%). Three patients had grade 4 thrombocytopenia and 2 had grade 4 neutropenia. Dose reduction was necessary in 3 patients and treatment cycle postponed in 2 patients because of hematological toxicity. Five patients (33%) needed G-CSF support. Two patients developed febrile neutropenia. No treatment-related deaths occurred. Gemcitabine was shown to be an active salvage regimen in patients with relapsed/refractory Hodgkin's lymphoma after ASCT, producing an overall response rate of 40%. Although, the median PFS time was short, some patients can be bridged to allogeneic SCT. Hematological toxicity was common.

Disclosures

Off Label Use: Gemcitabine in Hodgkin's Lymphoma.
A Combination of Lenalidomide and Rituximab (ReRi) As Salvage Therapy in Elderly Patients Affected By Diffuse Large B Cells (DLBCL) Lymphoma Relapsed and Refractory
Authors: Gini Guido , Bocci Caterina , Sampaolo Michela , Trappolini Silvia , Cacciagiù Sonia , Olivieri Jacopo , Olivieri Attilio , Offidani Massimo , Rupoli Serena , Poloni Antonella , Scortechini Anna Rita , Capelli Debora , Montanari Mauro , Scortechini Ilaria , Leoni Pietro
Published: November 2019

BACKGROUND

The incidence of non Hodgkin's lymphoma (NHL) increases with age, over one third of NHL cases involves elderly patients >70 years of age.

As a matter of fact, many elderly patients (pts) are not enrolled in controlled studies because they do not meet the inclusion criteria. This is the reason why the data from trials on elderly cases are not representative for the whole elderly population. Consequently, many of these patients do not benefit of new therapeutic progresses and the treatment is not yet adequate.

Moreover in case of relapse, salvage therapy in the elderly is virtually absent, and the prognosis is extremely poor.

Lenalidomide is an immunomodulatory drug with anti-angiogenetic and anti-neoplastic action on cancer cells and also has other anti-tumor activity by acting on the neoplastic microenvironment. Both monotherapy and combination of Lenalidomide with rituximab have shown efficacy in terms of overall response rate (ORR, Overall Response Rate) in the setting of salvage therapy in relapsed/refractory DLBCL, with an acceptable rate of hematologic and extra-hematological toxicities

PATIENTS AND METHODS

In the period 2013-2014 we consecutively treated 12 elderly patients affected by advanced DLBCL relapsed/refractory.

Median age at the start of treatment was 79 years (yrs) (62-86), 5 out 12 was over 80 yrs and 4 out 12 over 75 yrs. The median number of previous treatment was 3 (2-4), 3 pts were transplanted and 4 patients was refractory to previous line of therapy.

The treatment scheme included at first cycle: Rituximab 375 mg/m2 i.v. days 1, 8, 15, 22, Desametasone 5 mg p.o. days 1, 8, 15, 22 and Lenalidomide 15 mg/die p.o. from day 2 to 22. From the second to the sixth cycle we administered Rituximab 375 mg/m2 i.v. days 1, 14, Lenalidomide 20 mg/die p.o. from day 2 to 22

RESULTS

The overall response rate has been 75% (CR,PR,SD), 3 patients out 12 (25%) achieved a CR and 3 PR (25%).

The median PFS was 6 months and with a median follow up of 1 year the overall survival is 25%.(fig.1)

All deaths are due to lymphoma progression and the 3 patients in CR are still alive in CCR.

CONCLUSIONS

In this elderly and heavily pretreated group of patients our scheme containing Lenalidomide-Rituximab has shown an high rate of response with a good rate of CR and a promising trend in overall survival.

The treatment appears feasible and safe also in this particular group of frail patients and even if we need more data to confirm we think it will be possible to extend this therapy in frail elderly patients in a most precocious line of treatment.

Figure 1. Figure 1.

Disclosures

Offidani: Celgene, Janssen: Honoraria.
The BET Inhibitor OTX015 (MK-8628) Shows in Vivo Antitumor Activity in Combination with Additional Targeted Agents in Diffuse Large B-Cell Lymphoma (DLBCL)
Authors: Gaudio Eugenio , Cascione Luciano , Ponzoni Maurilio , Tarantelli Chiara , Bernasconi Elena , Riveiro Maria Eugenia , Cvitkovic Esteban , Zucca Emanuele , Bertoni Francesco
Published: November 2019

OTX015 (MK-8628) has shown anti-lymphoma activity in both the preclinical and clinical settings (Boi et al, CCR 2015; Stathis et al, EORT-NCI-AACR 2014). Here we report in vivo data on OTX015 in combination with four targeted compounds in DLBCL.

Methods. NOD-Scid (NOD.CB17-Prkdcscid/NCrHsd) mice were subcutaneously engrafted with the activated B-cell like DLBCL cell line SUDHL2 (15 x106 cells) and then divided into 10 groups (6 mice each). Treatment with OTX015 was started 3 days after the graft, and treatments with other drugs were initiated when mice developed palpable tumors (100 mm3). Tumor size was measured two times per week using digital calipers. Tumor volumes were calculated using the equation V = [length x width2]/2 (width and length are the shortest and the longest diameters of each tumor, respectively). Tumor specimens were collected at the end of treatment and necrosis was semi-quantitatively defined on the total amount of neoplastic tissue.

Results. Xenografts of SU-DHL-2 were treated with control or OTX015 (50 mg/kg once daily; QDx7/w x5w), the BTK-inhibitor ibrutinib (5 mg/kg; QDx2/w x5w), the mTOR-inhibitor everolimus (1 mg/kg, Qdx2/w x5w), the HDAC-inhibitor vorinostat (15 mg/kg; QDx2/w x5w), the anti-CD20 monoclonal antibody rituximab (3 mg/kg; QDx1/w x5w) as single agents or in OTX015-containing combinations. No weight loss was reported. OTX015, in accordance previously published data (Boi et al, CCR 2015), as single agent delayed tumor growth. Almost complete eradication of tumors was observed in mice treated with OTX015 combinations (p<0.001) (Figure 1). Three tumors for each group were H&E stained and necrosis percentage evaluated. Only control and vorinostat-treated tumors showed no or minimal (<5%) necrosis, which appeared higher in tumors treated with all the other drugs as single agents and in the combinations.

Conclusions. OTX015-containing combinations with everolimus, ibrutinib, vorinostat and rituximab showed very promising in vivo activity in an ABC-DLBCL model, providing the rationale for future clinical studies.

Figure 1. In vivo treatment of ABC-DLBCL SU-DHL-2 xenografts with MK-8228 as single agent and in combination with other targeted drugs. Figure 1. In vivo treatment of ABC-DLBCL SU-DHL-2 xenografts with MK-8228 as single agent and in combination with other targeted drugs.

Disclosures

Riveiro: Oncology Therapeutic Development: Employment. Cvitkovic:Oncology Therapeutic Development: Employment, Membership on an entity's Board of Directors or advisory committees. Bertoni:PIQUR Therapeutics AG: Research Funding; Oncology Therapeutic Development: Research Funding.
Establishment and Characterization of a Patient-Derived Model of ALK-Positive Anaplastic Large Cell Lymphoma
Authors: Wick Michael J , Helman Elena , Vaught Teresa L , Farley Monica , Rasco Drew W , Smith Lon , Tolcher Anthony , Patnaik Amita , Clark Michael , Papadopoulos Kyriakos P
Published: November 2019

Background: Anaplastic large cell lymphoma (ALCL) is an uncommon, aggressive CD30-positive T-cell lymphoma exhibiting or lacking chromosomal translocation involving the Anaplastic Lymphoma Kinase (ALK) gene and expression of ALK protein. ALK positive ALCL (ALK+-ALCL) typically has an improved prognosis compared with ALK-negative; however, patients often respond favorably to standard chemotherapy and CD30-directed immunoconjugates only to progress and are left with few treatment options. To help identify additional treatment options, we collected tumor tissue and established, characterized and evaluated in vivo an ALK+-ALCL patient-derived xenograft (PDX) model from a thirty one year old Caucasian female with relapsed ALCL following initial therapy with a pediatric S-phase specific regimen.

Methods: The ST2698 ALK+-ALCL model was established in CB17 SCID mice using tissue collected from a lymph node tumor biopsy. Clinical tissue, patient blood and the PDX model were subjected to WGS sequencing using an augmented and content-enhanced exome. The augmented exome is optimized to detect major cancer mutations by enhancing coverage over known sequencing gaps and GC-rich regions across >1300 cancer and 200 miRNA genes. We also performed whole-transcriptome sequencing on the PDX model. All data were analyzed using a cancer bioinformatics pipeline optimized for high accuracy detection of small variants and indels, somatic copy-number aberrations, gene expression and fusions. Drug sensitivity studies were performed evaluating sensitivity of the model to patient's current clinical treatment and relevant targeted therapies; study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion.

Results: Exome analysis identified several variants which were confirmed by transcriptome data. Efficacy studies confirmed model sensitivity to the patient's current clinical course (CHOP). In addition, ST2698 was found moderately sensitive to the IMiD lenalidomide (T/C=47%). However, treatment with the ALK-inhibitors crizotinib or ceritinib resulted in tumor regressions including durable complete responses in some mice.

Conclusion: We have established and characterized a patient-derived ALK+-ALCL xenograft model using DNA and RNA-based analysis. In addition we evaluated the model in vivo and confirmed sensitivity to the patient's current clinical course and identified two ALK inhibitors as active, including reported complete tumor regressions.

Disclosures

Rasco: Celgene: Research Funding; Asana BioSciences, LLC: Research Funding. Tolcher:Asana BioSciences, LLC: Consultancy, Research Funding; AbbVie: Consultancy; ArQule: Consultancy; Bayer: Consultancy; BioMed Valley Discoveries: Research Funding; Janssen R&D: Consultancy.
Ixazomib, an Oral Proteasome Inhibitor, Induces Rapid Mobilization of Hematopoietic Stem Cells in Mice
Authors: Ghobadi Armin , Rettig Michael P. , Holt Matthew , Ritchey Julie , Eissenberg Linda , DiPersio John F.
Published: November 2019

Introduction: Granulocyte colony-stimulating factor (G-CSF) is the most commonly used drug for stem cell mobilization. Unfortunately, 5-30% of patients fail to collect sufficient hematopoietic stem/progenitor cells (HSPCs) to proceed to transplant. New strategies are needed to increase HSPCs collection in these patients. We previously reported that bortezomib directly and rapidly mobilizes HSPCs in mice by modulating the VLA-4/VCAM-1 axis (Ghobadi A et al. Blood 2014, 124: 2742-2753). Ixazomib (MLN9708) is a next-generation small-molecule proteasome inhibitor that has several potential advantages over bortezomib including oral route of administration. Here we study the effect of ixazomib on stem cell mobilization in mice.

Methods: DBA mice were treated with: (1) ixazomib via oral gavage (o.g., 8 mg/kg), (2) a single intravenous (iv) dose of bortezomib (0.8 mg/kg), (3) AMD3100 (5 mg/kg) SC, and (4) control diluent for ixazomib (2-hydroxypropyl-β-cyclodextrin [HPBCD]). Blood was harvested at baseline and 12, 15, and 24 hours (h) after ixazomib and bortezomib administration and at 3 hours and 12 hours after AMD3100 administration. Harvested peripheral blood (PB) was plated on MethoCult media (Stem Cell Technologies) for colony forming unit-cells (CFU-C) enumeration.

Results: Compared to vehicle (ixazomib diluent, 2-hydroxypropyl-β-cyclodextrin [HPBCD]), ixazomib mobilized significantly more peripheral blood colony forming unit-cells (CFU-C) at 12 - 15 h after administration (mean peak CFU-C: 105/ml vs. 870/ml respectively, i.e., a 0.6 vs. 5-fold increase in CFU-C , P < 0.02; Figure 1A). The majority of CFU-Cs mobilized by ixazomib were CFU-GM (CFU-granulocyte and monocyte, Figure 1B). The magnitude and kinetics of HSPC mobilization in the single ixazomib oral gavage group was identical to a single dose of IV bortezomib (Figure 1C, mean peak of 870/ml vs. 975/ml respectively, P > 0.66). There was no statistically significant difference in peak HSPC mobilization between the ixazomib (12 h after ixazomib administration) and AMD3100 groups (3 h after AMD3100 administration) (mean peak of 870/ml vs. 1240/ml respectively, P = 0.36) suggesting that ixazomib is a potent HSPC mobilizing agent (Figure 1D) with intermediate mobilization kinetics compared to AMD3100 (2-3 hours) and G-CSF (5-6 days) and with identical kinetics to IV bortezomib (12-15 hours).

Conclusion: Ixazomib is a potent and modestly rapid HSPC mobilizer agent in mice. Kinetics and magnitude of HSPC mobilization by ixazomib is identical to bortezomib. Trials to assess proteasome inhibitors as mobilizing agents are currently underway at Washington University.

Disclosures

No relevant conflicts of interest to declare.
Correlation Between Tumor Microenvironment and the Inflammatory Factors in Lymphoma-Burdened Mice
Authors: Zhang Jun , Ni Jun , Xu Pei , Shi Qingqing , Gu Jian , Depei Wu
Published: November 2019

Introduction

Since Vichow proposed the hypothesis that chronic inflammation was closely related to tumor in 1863, experimental, clinical and epidemiological studies have shown that inflammation plays an important role in tumor progression, immune response, tumor therapy and prognosis. In this study, 38B9 cells were injected into BALB/c mice to establish lymphoma-burdened mice model. The expression of IL-10, TNF- and IL-6 were observed in plasma and tissues to study the effects of tumor inflammatory factors on tumor environment.

Methods

Ten 9-week-old BALB/c mice were randomly divided into the control group and the tumor-burdened group. Each mouse in tumor-burdened group was injected with 2×106/200μl 38B9 cells, while the control group was injected with 200μl PBS buffer solution. The establishment of B-cell lymphoma tumor-burdened mice model was confirmed by magnetic resonance information (MRI). The peripheral blood samples were collected before injection, after tumorigenesis and when sacrificed. The tumor tissues were also collected. The level of IL-6, IL-10, TNF-α in the plasm were detected by enzyme-linked immuno sorbent assay (ELISA) and the relative value of mRNA in tissues were measured by quantitative real-time polymerase chain reaction (qRT-PCR).

Results

1.Compared with the control group, the levels of IL-6 and TNF-α in the tumor-burdened group were increased after injected 14 days (P <0.05). However, the level of IL-10 showed no significant difference (P >0.05). After injected 19 days, the levels of IL-6, IL-10 and TNF-α of tumor-burdened group were decreased (P<0.05).

2. The expression of IL-10 mRNA in tumor tissues was lower than the control group (P <0.05), while the expression of IL-6 mRNA in borderline tissues was higher than the control group (P<0.05). Compared with tumor tissue, the expression of IL-10 mRNA was increased in borderline tissue (P <0.05). The level of IL-6 and TNF-α showed no significant difference (P>0.05).

Conclusions

1. The level of IL-6, IL-10 and TNF-α in the tumor tissues and plasma were significantly higher after tumorigenesis.

2. The increased expression of inflammatory factors is related to tumor-induced inflammatory microenvironment.

Disclosures

No relevant conflicts of interest to declare.
Modulation of Leukotriene Signaling Affecting Cell Growth in Chronic Myeloid Leukemia: A Key Pathway to Cure?
Authors: Yektaei Elham , Nilsson Anders , Näsman-Glaser Barbro , Ekblom Marja , Qian Hong , Rådmark Olof , Zovko Ana , Kanter Lena , Wallvik Jonas , Stenke Leif
Published: November 2019

Background: Recent data suggest that tyrosine kinase inhibitor (TKI)-insensitive leukemic stem cells often prevail in chronic myeloid leukemia (CML) patients subjected to long-term TKI treatment. To achieve cure additional aberrant pathways in these patients, such as leukotriene (LT) signaling, may need to be targeted. We have previously shown that human CML cells have an increased capacity to produce LT and that LT can stimulate normal myelopoiesis (Tornhamre ExpHem 2003). Chen later noted a striking up-regulation of ALOX5/5LO (catalyzing an initial step in LT formation) in CML mice, and that inhibition of this enzyme improved survival of these animals by a magnitude similar to that induced by imatinib (Chen NatureGen 2009). Here wexve assessed the effect of LT modulating agents on the growth of human CML cells in vitro.

Materials and methods: Human CML cells, derived from the three cell lines K562, Kcl22 and KU812, were grown in microtitre plate cultures, in the presence of RPMI 1640. The MTT technique was used to evaluate the number of viable cells at 72 hours. Protein expression was assessed by Western blot and immunocytochemistry.

Results: Several specific modulators of LT-signaling were capable of inducing dose-dependent growth inhibitory effects on CML cells. Thus, the cysteinyl-LT receptor antagonist montelukast, a drug with approved clinical use in human asthma, was shown to reduce the growth of all three tested CML cell lines. In Fig. 1 this is examplified by K562 cells, where also additive effects between montelukast and imatinib are indicated. All cell lines expressed the cysLT1-receptor. Furthermore, the LTB4 receptor antagonist etalocib, as well as the 5-LO activating protein (FLAP) inhibitors licofelone and Bay-X-1005, also executed inhibitory effects at concentrations considered as physiological. As expected, addition of the TKIs imatinib, nilotinib or dasatinib to the cultures also generated dose-dependent inhibitory effects on the growth of all tested CML cell lines. An LD50 of approximately 0.5 µM, 10 nM and 0.1 nM was noted for imatinib, nilotinib and dasatinib, respectively, for K562 cells at 72 hours.

Conclusions: We demonstrate that several compounds, known to specifically inhibit leukotriene signaling by different mechanisms, were at clinically relevant concentrations capable of significantly suppressing the growth of human CML cells. Some of these compounds are already in clinical practice for non-hematological disorders. They could provide an additional therapeutic possibility in the quest to cure CML.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
BCR-ABL Fluctuation Analysis in Chronic Phase Chronic Myeloid Leukemia (CP-CML) Patients with MR 4.5
Authors: Pereira Mariana Pinto , Vanelli Tito , Gomes da Silva Eduardo , Dias Américo André , Burin Mariana M , Soares Tahiane , Silla Lucia , Daudt Liane , Fogliatto Laura
Published: November 2019

Introduction: Imatinibe, a target tyrosine kinase inhibitor, have revolutionized CP-CML treatment. Considering that it is being discussed imatinibe treatment interruption in patients with MR ≥ 4.5 lasting two years or more, we believe to be important to establish predictors of major molecular response (MMR) loss. Fluctuations in BCR-ABL transcripts are being advocated to be able to predict MMR loss, although the real impact of small fluctuations among patients with in MR ≥ 4.5 is uncertain.

Objective: Correlate BCR-ABL fluctuations and MMR loss in patients considered to be in MR ≥ 4.5, as well as to recognize possible factors associated with these small fluctuations.

Methods: We conducted a retrospective analysis in CP-CML patients receiving imatinibe (as first or second treatment line) that achieved MR ≥ 4.5 (defined as a 4.5 log reduction on an international scale) and surveyed for BCR-ABL fluctuations. We considered a fluctuation to be a rise of at least 0.5 log of BCR-ABL transcripts. Treatment interruptions were considered only when imatinibe was not taken for 15 or more consecutive days. The presence of comorbities was evaluated by applying the Charlson Index.

Results: Fifty-eight patients were evaluated between with a median follow-up 7 years. Fifty-five percent were men and 28 were women, with a median age of 46 (18 - 93) years-old, most of the patients were older than 60 yo (75,9%). Among 32 patients evaluated by the Sokal risk score 97,5% were estimated to be low or intermediate. Twenty six patients presented at least one fluctuation. Regarding the number of fluctuations per patient, 18 presented only 1 fluctuation, 5 presented 2 fluctuations and 3 patients presented 3 fluctuations. Among those patients with at least one fluctuation 3 (11%) presented MMR loss, while there was none among patients without documented fluctuations (p=0,15). Among these three patients considered to have MMR loss, two presented fluctuations that reached MR 3.0, while the other patient presented a history of long treatment suspension. More patients that interrupted treatment presented BCR-ABL fluctuations, although this difference was not statically significant (78% vs 50% p=0,11).

Conclusion: Small fluctuations among patients with MR ≥ 4.5 didn't seem to correlate with MMR loss, however we do believe that our population is underpowered to rule out this difference. We considered that it very interesting that patients which reached MR 3.0 presented MMR loss (considering that the other patient had to abandon treatment during pregnancy).

Disclosures

No relevant conflicts of interest to declare.
Predictive Factors of Treatment Response to Hypomethylating Agents in Patients with Myelodysplastic Syndrome
Authors: Wu Ping , Du Xin , Weng Jianyu , Deng Chengxin , Geng Suxia , Lu Zesheng , Lai Peilong , Huang Xin , Luo Chengwei
Published: November 2019

Background: Hypomethylating agents (HMAs), including decitabine and azacitidine, have improved outcomes for patients with myelodysplastic syndrome (MDS). However, not all patients benefit from this therapy, and no reliable prognostic tool can predict differential likelihood of benefit from HMAs.

Methods: To investigate predictive factors of treatment response to HMAs in patients with MDS, we analyzed the baseline biomarkers, clinical variables and TP53 mutations of 52 newly diagnosed Chinese patients with MDS who received decitabine or azacitidine (1-23 cycles, median number cycles: 7)

Results: In the 46 assessable patients who received more than 2 cycles of standard treatment, a total of 22 patients had an overall response after treatment, and 24 patents showed no response. The results showed that there was no significant difference in response by treatment regimen (P=0.147) or source of sample (P=0.413). Furthermore, age (P=0.382), sex (P=0.595), IPSS risk groups (P=0.117) and cytogenetic abnormalities (P=0.935) were not associated with response rate. However, in univariate analysis of the overall cohort (n=40) who received more than 3 cycles of standard treatment, the response rate was significantly higher for those patients who had platelet count doubling after 2 cycle of treatment (P=0.013), but there was no statistically difference after 1 cycle(P=0.376). Moreover, a total of 5 patients (11%) had TP53 mutations, overall response rate to HMAs was 100% in patients with TP53 mutation, which is significantly higher than that of patients with wild type cases (p = 0.013).

Conclusions: These findings indicate that patients with TP53 mutation or achieving platelet count doubling after 2 cycle of HMAs respond well.

Disclosures

No relevant conflicts of interest to declare.
A Rapid, Sensitive and Specific Method for Quantifying Calr Mutant Allele Burden in Persons with Myeloproliferative Neoplasms
Authors: Zhou Jiao , Yao Qiumei , Gale Robert Peter , Li Jinlan , Li Lingdi , Li Ning , Chen Shanshan , Ruan Guorui
Published: November 2019

Background: CALR mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2V617F. Consequently rapid, sensitive and specific methods to detect and quantify these mutations are needed.

Methods: We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2V617F negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden.

Results: We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5-5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5-5%) in a wild-type background.

Conclusions: PCR amplification followed by fragment length analysis is a rapid, sensitive and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PV with JAK2V617F and for estimating mutant allele burden.

Figure 1. Standard curve for the detection of mutant allele burden. Figure 1. Standard curve for the detection of mutant allele burden. Figure 2. Titration analyses of sensitivity of CALR mutation screening by sequencing and fragment analyses. Figure 2. Titration analyses of sensitivity of CALR mutation screening by sequencing and fragment analyses. Figure 3. Figure 3 Sequencing traces show heterozygous mutation of CALR. Gene scan electropherogram from PCR method and partial sequence of CALR exon 9 from sequencing method (numbering according to GenBank access number: NC_000019.9). A-P: Detected a wild type and 15 CALR mutation types by sequencing and fragment analysis methods. A: wild type, B-I: deletions, H-L: insertions, M-P: complex indels. Figure 3. Figure 3 Sequencing traces show heterozygous mutation of CALR. Gene scan electropherogram from PCR method and partial sequence of CALR exon 9 from sequencing method (numbering according to GenBank access number: NC_000019.9). A-P: Detected a wild type and 15 CALR mutation types by sequencing and fragment analysis methods. A: wild type, B-I: deletions, H-L: insertions, M-P: complex indels.

Disclosures

No relevant conflicts of interest to declare.
GDF11 Increased in Patients with Myelodysplastic Syndrome
Authors: Han Yu , Wang Huaquan , Shao Zonghong
Published: November 2019

Objective To analyze the concentration of growth differentiation factor 11(GDF11) in peripheral blood of patients with myelodysplastic syndrome (MDS), so as to evaluate the relationships between these changes and erythropoiesis functions and to explore the role of GDF11 in the pathogenesis of MDS.

Methods The concentration of GDF 11 in peripheral blood was detected by enzyme-linked immuno sorbent assay in 44 MDS patients and 10 normal controls from September 2014 to June 2015 at our hospital. The percentage of nucleated erythrocyte (CD235a) in bone marrow was detected by flow cytometry. The correlation between these changes and erythropoiesis functions, including red blood cell count, hemoglobin, reticulocyte (RET%), hematokrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular-hemoglobin concentration (MCHC) and late erythroblast in bone marrow were evaluated.

Results (1)The concentration of GDF11(128.67±47.62)in high-risk MDS patients was significantly higher than that of low-risk MDS patients (65.96±36.55,p<0.01)and higher than that of normal controls (29.76±10.10,p<0.01); The concentration of GDF11 in low-risk MDS patients was significantly higher than that of normal controls (p<0.05). (2) The expression of CD235a in high-risk group(38.49±5.42)was not different with that in low-risk group(42.64±7.36, p>0.05). (3)In high-risk MDS patients, the expression of GDF11 was negatively correlated with Hb, RET%, RBC, MCHC, Hct in peripheral blood and late erythroblast, CD235a+ cells in bone marrow(r=-0.437,r=-0.428,r=-0.444,r=-0.553,r=-0.661,r=-0.436,r=-0.52,all p<0.05),and the expression of GDF11 was positively correlated with MCV(r=0.52, p <0.05),but it was not correlated with MCH (p >0.05).(4) In low-risk MDS patients, the expression of GDF11 was negatively correlated with Hb, RET% (r=-0.491Ar=-0.606,both p<0.05),it was not correlated with RBC, MCHC, MCV, MCH, Hct, late erythroblast and CD235a+ cells (all p>0.05).

Conclusion GDF11 increased in patients with MDS and it was negatively correlated with late erythropoiesis.

Disclosures

No relevant conflicts of interest to declare.
IGF2BP1 Protein Binding of Erythroblast Transcription Factor mRNA: A Cytoplasm-Localized Mechanism for Globin Gene Regulation
Authors: de Vasconcellos Jaira F. , Lee Y. Terry , Byrnes Colleen , Clarke Benjamin A. , Xu Pauline C. , Rabel Antoinette , Proia Richard L. , Miller Jeffery L.
Published: November 2019

Pharmacologic and genetic regulation of fetal hemoglobin (HbF) remains a major goal for treatment of the beta-thalassemias and sickle cell disease. Recently, the let-7 microRNA heterochronic pathway of RNA-binding factors was shown to be developmentally correlated with HbF expression in humans. Experimental manipulation of the pathway components (LIN28, let-7, and the IGF2 binding proteins) demonstrated moderate increases in fetal hemoglobin (around 15-35% HbF) with the exception of IGF2BP1, which caused a nearly complete reversal in gamma- and beta-globin gene expression resulting in HbF levels of 60-70% among cultured adult human erythrocytes. To further explore IGF2BP1 effects on HbF and erythropoiesis, lentiviral transduction of CD34(+) cells was performed with let-7 resistant expression of IGF2BP1 driven by the human SPTA1 gene (IGF2BP1-OE). Donor-matched control transductions were studied for comparison. For protein localization studies, confocal imaging of sorted cells was utilized. IGF2BP1-OE caused IGF2BP1 protein expression throughout the cytoplasm and localized to small granules. Granules were unevenly distributed and more abundantly observed in the perinuclear regions. Nuclear detection of IGF2BP1 protein remained at background control levels. We thus hypothesized that IGF2BP1 protein in these cytoplasmic granules may regulate globin gene expression by binding RNAs that encode epigenetic modifiers of the beta-globin locus. To test this hypothesis, RNA-immunoprecipitation (RIP) was performed using a RIP-certified anti-IGF2BP1 antibody. Next generation sequencing and qRT-PCR were used to determine the mRNA species bound by IGF2BP1. RIP-enrichment of BCL11A mRNA was identified (4.6 ± 1.2 fold compared to input sample), as well as mRNA encoding other modifiers of globin gene expression including KLF1 and ZBTB7A. Further analyses of BCL11A showed no significant changes in BCL11A mRNA levels among IGF2BP1-OE cells (control: 6.5.E+03 ± 3.8.E+03, IGF2BP1-OE: 4.1.E+03 ± 1.2.E+02, p=0.403). However, BCL11A protein detection was reduced to background levels by Western analysis compared to transduction control cell lysates. These studies suggest that RNA-binding and cytoplasmic compartmentalization provide IGF2BP1 with a mechanism for increasing fetal hemoglobin via post-transcriptional regulation of globin gene transcription factors.

Disclosures

No relevant conflicts of interest to declare.
Intravascular Large B Cell Lymphoma in the United States (US): A Population- Based Study Using Surveillance, Epidemiology, and End Results Program and National Cancer Data Base
Authors: Rajyaguru Devalkumar J , Bhaskar Chaithanya , Borgert Andrew J , Smith Angela , Parsons Benjamin
Published: November 2019

Background: Intravascular large B cell lymphoma (IVLBCL) is a rare and aggressive non-Hodgkin lymphoma lacking any prior US based population studies. We aimed to conduct a population based study of IVLBCL in the US and compare characteristics and survival to diffuse large B cell lymphoma, not otherwise specified (DLBCL, NOS).

Methods: We used Surveillance, Epidemiology, and End Results (SEER) registries from 2000 to 2013 to calculate incidence rates (case/1,000,000) and age-adjusted to the US 2000 standard population. We collected patient level data from the NCDB Participant User Files using ICD-O-3 histology code 9680 with ICD-O-3 primary site modifier C499 and analyzed for demographic and clinical characteristics as well as overall survival (2004-2013). Chi-Square test was used to compare the characteristics of patients with IVLBCL to that of DLBCL, NOS. Overall survival analysis was performed using Kaplan- Meier method and compared by log rank. Overall survival (OS) of IVLBCL was compared to propensity-matched cohort of DLBCL, NOS. Multivariate analysis was conducted using weighted Cox Proportional Hazard regression model.

Results: In the years 2000-2013, the overall crude incidence rate of IVLBCL was 0.093 (case/1,000,000) and age-adjusted incidence rate of IVLBCL was 0.095 (case/1,000,000). A total of 388 IVLBCL patients were identified in NCDB database. Patients with IVLBCL (n=388), compared to DLBCL, NOS (n=155,436), were more likely to be of age ≥ 65 (64.2% vs 56.9%, p=0.0039), female (51.8% vs 46.5, p=0.036), diagnosed at advanced stage (stage IV, 51.5% vs 33.4%, p<0.0001) and less likely to receive chemotherapy (70.4% vs 78.9%, p = 0.0001).The median 1, 3 and 5-year OS rates for IVLBCL were 66.2%, 51.8% and 46.3% respectively. After propensity matching, the 5-year overall survival of IVLBCL was comparable to DLBCL, NOS (46.6% vs 45.4%, p=0.61). On multivariate analysis, advanced age (HR 1.03, 95% CI 1.032-1.034; p<0.0001), advanced stage (Stage III: HR 1.34, 95% CI 1.31-1.38; <0.0001, Stage IV: HR 1.81, 95% CI 1.77-1.85; p<0.0001), having Medicaid (HR 1.72, 95% CI 1.66-1.79; p<0.001) or Medicare (HR 1.24, 95% CI 1.20-1.26; p<0.0001) predicted worse OS, whereas female gender (HR 0.86, 95% CI 0.85-0.88; p<0.0001) and use of radiation therapy (HR 0.76, 95% CI 0.75-0.78; p<0.0001) predicted better OS.

Conclusions: This is the largest series of IVLBCL reported to date. Our study provides characteristics and OS of a large cohort of this rare entity, which can be used for patient education and may provide background information for future research. In the Rituximab era, the OS of patients with IVLBCL is comparable to DLBCL, NOS. In addition, our study also reports predictors of OS in patients with IVLBCL.

Disclosures

No relevant conflicts of interest to declare.
An Essential Erythroid-Specific Enhancer of ATP2B4 Associated with Red Blood Cell Traits and Malaria Susceptibility
Authors: Stern Emily N , Lessard Samuel , Schupp Patrick G. , Sher Falak , Lettre Guillaume , Bauer Daniel E.
Published: November 2019

Genome-wide association studies (GWASs) have identified thousands of common genetic variants associated with human traits and disease susceptibility. Given that the majority of GWAS-identified SNPs are located within non-coding regions of the genome, the mechanisms of these trait associations are frequently unknown. Here we investigate the biological underpinnings of common trait-associated genetic variation at ATP2B4, a gene on chromosome 1q32 that encodes a major plasma membrane calcium ATPase (also known as PMCA4). Genetic variation within this locus is associated with several phenotypes including various red blood cell traits (mean corpuscular hemoglobin concentration and red cell distribution width) as well as susceptibility to severe malaria infection. We conducted an expression quantitative trait loci (eQTL) mapping analysis in human erythroblasts and identified a set of SNPs associated with ATP2B4 gene expression in cis. These included the same genetic variants found by GWAS to be associated with RBC traits and malaria susceptibility. Furthermore, these SNPs overlap an intronic erythroid DNase I hypersensitive site at ATP2B4. An analysis of the ENCODE database showed that this element was erythroid specific in that it lacked DNase I hypersensitivity in >30 queried non-erythroid cell types. We used the CRISPR/Cas9 genome editing system to delete this element in HUDEP-2 immortalized human erythroid precursor cells. We observed that cells bearing a 927 bp biallelic deletion of this noncoding element displayed near complete loss of ATP2B4 expression (3% residual gene expression), while cells bearing heterozygous deletions showed an intermediate gene expression phenotype. We identified a core element that encompassed 3 of the highly trait associated SNPs and 5 GATA1-binding motifs. Biallelic deletion of this 98 bp core led to 83% reduction in ATP2B4 expression. Disruption of individual GATA1-binding motifs resulted in partial reduction of gene expression, suggesting the contribution of multiple binding sites to appropriate gene expression. Overall, this study suggests that variation within an essential erythroid-specific enhancer of ATP2B4 underlies the association of ATP2B4 with RBC traits and malaria susceptibility. Furthermore these results encourage combined analyses of gene expression, chromatin state, and prospective genetic perturbation as a means to explore the variants, elements and genes responsible for heritable blood phenotypes.

Disclosures

No relevant conflicts of interest to declare.
A Pilot Intervention to Improve Hydroxyurea Adherence in Youth with Sickle Cell Disease through Community Health Workers: The "Habit" Study
Authors: Green Nancy S. , Manwani Deepa , Soto Luisa , Castillo Yina , Ireland Karen , Jin Zhezhen , Smaldone Arlene
Published: November 2019

Background: Chronic disease regimens for youth require self-management skills for a daily medication habit. Incomplete adherence to hydroxyurea (HU) is a treatment barrier for sickle cell disease (SCD) (Walsh KE, Pediatrics 2014). HU induction of fetal hemoglobin (HbF) is dose-dependent but not a standardized target (Ware RE, Hematology Am Soc Hematol Educ Program, 2015). In a previous 2-site retrospective analysis of youth with SCD, large deviation from historical personal best (PBest) HbF, a clinic-based form of maximum dose (Green NS, PBC, 2016), was common and correlated with increased acute hospital use. Here we hypothesized that: 1) A HU habit can develop through education and support of youth-parent dyads from a structured intervention of community health workers (CHW), augmented by text messaging; 2) Improved used will increase HbF; 3) The intervention is feasible and acceptable.

Methods: We conducted a 2-site R21-funded randomized controlled feasibility trial (RCT), "Hydroxyurea Adherence for Personal Best in Sickle Cell Treatment: HABIT" (Smaldone A, Contemp Clin Trials, 2016)." Youth- parent dyads (youth ages 10-18) were recruited for the 6-month intervention. Eligibility required a ≥15% drop from PBest HbF on stable HU dose over the preceding year. Intervention dyads received a 6-month structured intervention of education and support by CHWs with home visits and text messaging, versus educational materials. Primary outcomes were HbF, % deviation from PBest HbF and HU pharmacy refills.

Results: Using 2:1 randomization, 28 parent-youth dyads participated (18 intervention, 10 control): mean age 14.3±2.6 years, 42.9% female. At baseline, no differences were seen in HbF (10.5% vs. 13.4%, p=0.25), but deviation from PBest HbF was higher in the control group (-42.6%) than the intervention (18.1%, p=0.009). Using a growth model for % deviation from PBest HbF, the intervention group demonstrated a modest absolute improvement in deviation from PBest (2.2% per month higher in the intervention group), peaking at month 4 (p=0.31), and modest gain in mean HbF (3 versus 1.8). Four subjects exceeded their PBest. Compared to the year before intervention, pharmacy refills improved for the intervention group: 64.6% year prior, 80.7% (months 0 to 3), 85.9% (months >3 to 6), while the control group was unchanged (79.2% year prior, 83.6% months 0 to 3, 79.4% months >3 to 6). Intervention feasibility and acceptability by dyads were excellent.

Conclusions: The 6-month pilot RCT "HABIT" may improve HU adherence measured by improved HbF, deviation from PBest and pharmacy refill, was feasible and acceptable, and suggests utility as an intervention. Based on these findings, a multi-center HABIT RCT of 1-year duration powered to test efficacy is planned.

Funded by 5R21NR013745 (PIs Green, Smaldone)

Figure Figure.

Disclosures

No relevant conflicts of interest to declare.
Early Renal Disease in Children with Sickle Cell Disease from Malawi
Authors: Heimlich Jonathan Brett , Chipoka Godwin , Ellis Graham , Elsherif Laila , David Emeraghi , Kamthunzi Portia , Krysiak Robert , Majawa Yacinta , Mafunga Pilirani , Zhou Qingning , Cai Jianwen , Key Nigel S. , Gopal Satish , Ataga Kenneth I.
Published: November 2019

Sickle cell nephropathy (SCN) is a prevalent complication among adults with sickle cell disease (SCD) and has been observed in younger populations, suggesting potential early renal involvement in pediatric patients. Initial hyperfiltration and albuminuria followed by frank proteinuria, leading to declining GFR and eventual end stage renal disease is assumed to be the typical progression of SCN; however, few clinical biomarkers exist to identify early-stage renal disease. We describe the renal profile in 119 children with SCD at Kamuzu Central Hospital in Lilongwe, Malawi and propose a novel urinary biomarker for the identification of children with early renal disease. Among children with confirmed HbSS disease (females 47.9%; median age 9.0 years, IQR: 5, 11), 21.6% were found to have a urinary albumin to creatinine ratio (UACR) 30mg/g or above. Baseline laboratory and clinical parameters stratified by UACR are shown in Tables 1 and 2. Patients with increased levels of UACR were found to be significantly older, and have significantly elevated plasma levels of lactate dehydrogenase (LDH), total bilirubin, and indirect bilirubin when compared to those without albuminuria (p<0.05). No association was seen between albuminuria and either hemoglobin or plasma hemoglobin. Albuminuria was also significantly associated with elevated levels of nephrin, a urinary marker of glomerular injury (p<0.01). Multivariable logistic regression was used to investigate risk factors that are associated with albuminuria (UACR >= 30). Urine nephrin was significantly associated with albuminuria (regression coefficient estimate: 0.00188, SE: 0.000571, p = 0.0010). Additional analysis using a nephrin cut-point of 293 pg/mL, the median value in the cohort, revealed a 17.8 times greater odds of having albuminuria in children with nephrinuria above this value. These data taken together suggests that a significant proportion of children with SCD in Malawi exhibit renal involvement early in life and could be at risk for worsening nephropathy and end-stage renal disease as they grow older. Our data further indicates that urinary nephrin could be utilized as an early marker of glomerular disease in SCN and possibly prompt earlier intervention in these children. The discordant association of albuminuria with clinical markers of hemolysis suggests that hemolysis may not play a substantial role in the pathogenesis of albuminuria in this population. Increased surveillance of children with SCD for renal complications can ultimately inform management strategies to improve outcomes and increase life expectancy among children with SCD.

Disclosures

No relevant conflicts of interest to declare.
Termination and Frameshift Mutations in ELANE Are Associated with Adverse Outcomes in Patients with Severe Chronic Neutropenia
Authors: Dale David C. , Makaryan Vahagn , Kelley Merideth L. , Bolyard Audrey Anna , Boxer Laurence A. , Newburger Peter E. , Bonilla Mary Ann , Klimiankou Maksim , Skokowa Julia , Zeidler Cornelia , Welte Karl
Published: November 2019

Background: Mutations in ELANE are the most common cause for both cyclic and severe congenital neutropenia. We previously reported that some mutations, e.g., G214R and C151Y, are associated with more severe outcomes. (Makaryan, Zeidler, et al; Curr Op Hematol. 2015;22:3-11) We also found that termination and frameshift mutations are also associated with a high risk of MDS/AML, death from infection or need for transplantation. To aid in clinical care of SCN patients, we compared the clinical characteristics of patients with termination and frameshift who have or have not developed MDS/AML.

Methods: We reviewed clinical records for 403 patients with severe chronic neutropenia and mutations in ELANE. We identified 44 patients with termination or frameshift mutations, both categories predicted to lead to synthesis of mutant neutrophil elastase that is missing a number of terminal amino acids and causing distortion of the three dimensional structure of the protein.

Results:There are currently 11 cases of MDS/AML in the 44 patients with termination or frameshift mutations. Table 1 compares clinical characteristics of these two groups.

There was no significant relationship of baseline ANC values, G-CSF dose or duration of G-CSF treatment. There appeared to be a higher risk of AML for the female patients, but the difference was also not significant.

The leukemia cases occurred with mutations in exon 3 (1), exon 4 (1) and exon 5 (9). The mutations in the group without MDS/AML were in exon 4 (8) and exon 5 (25). To date, there have been six distinct frameshift mutations associated with MDS/AML. There have been 3 distinct termination mutations associated with MDS/AML; three unrelated patients have the identical mutation C223ter. All of the patients in the SCNIR database who have this specific mutation have developed MDS/AML.

Conclusion: Frameshift and termination mutations appear to convey a high risk of MDS/AML in patients with severe chronic neutropenia. We believe patients with these mutations should be on a "watch list" with particular attention to surveillance and regular bone marrow examinations. Mutation C223ter, together with G214R and C151Y, appear to be particularly high risk. We recommend that all patients with these mutations be considered for early HSCT.

Disclosures

Dale: Amgen: Consultancy, Honoraria, Research Funding. Boxer:Amgen: Equity Ownership.