bluuebunny/crossref_metadata_2025 huggingface dataset:
Sezary Syndrome: Clinicopathological and Immunophenotypical Characteristics and Their Impact On Survival
Authors: Deaver Darcie , Horna Pedro , Sokol Lubomir
Published: October 2019

Abstract 5085

Background:

Sezary syndrome is a rare leukemic subtype of cutaneous T cell lymphoma with aggressive clinical behavior and poor prognosis compare to the most common type of CTCL, mycosis fungoides. Hematologic criteria for the diagnosis of Sezary syndrome include an absolute Sezary cell count of >1000cells/mm3 in peripheral blood the presence of a clonal T cell population, increased CD4/CD8 ratio, and aberrant expression of T cell markers.

Objectives:

To evaluate clinical, pathological and immunophenotypical characteristic in patients with Sezary syndrome and determine the impact of these factors on disease survival.

Research Design and Methods:

Retrospective chart review of 17 consecutive patients diagnosed with Sezary syndrome at Moffitt Cancer Center from January 1998-June 2012. Data points collected were age, gender, stage of disease, date of diagnosis, date of death, and analysis of peripheral blood by flow cytometry. Statistical analyses were performed by SPSS statistical software (IBM, Somers, NY). All causes of death were included in the survival analysis. Survival curves were estimated by the Kaplan-Meier method.

Results:

Of the 17 patients evaluated 10 (58. 8%) were female, and 7 (41. 2%) were male, median age was 71 years (range 50–89). Patients were staged according to 2007 ISCL/EORTC staging system 8 (47. 1%) stage III and 9 (52. 9%) stage IV. Median survival was 20. 9 months (range 0–107. 6) for all stages; 27 months and 9. 36 months for stage III and IV, respectively. As expected, stage IV disease was associated with a higher CD4:CD8 ratio (median 62. 5/uL versus 8. 6/uL), absolute lymphocyte count (median 9, 860/ul versus 2800), and absolute Sezary cell count (median of 8, 700/uL versus 1, 700/uL). Interestingly, stage IV disease was also associated with a higher absolute NK cell count (median 280/uL versus 60/uL) and a lower absolute CD8 T cell count (median 165/uL versus 290/uL). Of all parameters studied the only ones showing statistical correlation with the stage of disease were the NK cell count (p = 0. 018) and the CD4:CD8 ratio (p = 0. 039). The immunophenotype of the Sezary cells did not correlate with stage of disease. Survival analysis did not show any significant differences by grouping patients according to high or low levels of variables described above, although the series is small.

Conclusion:

We concluded that increased burden of disease in the peripheral blood did not affect overall survival in our patient population. However, Higher CD4:CD8 ratio, higher absolute lymphocyte count, and lower absolute CD8+ T cell count was associated with more advanced stage of disease suggesting that a lack of cytotoxic T cells can be responsible for profound immunosuppression and disease progression found in patients with advanced stage of CTCL.

Disclosures:

No relevant conflicts of interest to declare.
The Incidence, Characteristics, and Outcomes of Venous Thromboembolism in Patients with Follicular Lymphoma Compared to Diffuse Large B-Cell Lymphoma
Authors: Wang Tzu-Fei , Liu Weijian , Carson Kenneth R.
Published: October 2019

Abstract 5090

Background:

Previous studies have demonstrated a widely variable incidence of thromboembolism (VTE) in non-Hodgkin lymphoma (NHL) patients, ranging from 3% to 60%. This is in part because many previous studies have considered NHL as a single disease entity, combining a heterogeneous group of histological subtypes, each with a potentially different thrombogenic potential. We sought to clarify differences in VTE incidence between the indolent and intermediate grade forms of NHL, by looking at follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), respectively. Furthermore, since chemotherapy is a major risk factor for VTE, we compared VTE incidence rate in the pre- and post-chemotherapy time periods.

Patients/Methods:

We assembled a retrospective cohort of the United States veterans diagnosed with DLBCL or FL between 1998 and 2009. VTE was identified by ICD-9 codes, while DLBCL or FL was identified by ICD-O3 codes in the Veterans Health Administration central cancer registry. Patients who did not receive any chemotherapy or had a history of VTE prior to the diagnosis of lymphoma were excluded. VTE rate was compared by Chi-square testing.

Results:

A total of 2094 DLBCL patients and 470 FL patients were included. In the period between lymphoma diagnosis and treatment initiation, VTE occurred at a rate of 18. 0 per 100 person-years in DLBCL and 2. 3 per 100 person-years in FL (p<0. 0001). Similarly, in the 6 months following treatment initiation, DLBCL patients had a VTE rate of 12. 3 per 100 person-years, compared to 7. 3 per 100 person-years in FL (p=0. 039). In DLBCL, patients had a higher risk of VTE between diagnosis and treatment compared to the 6 months after treatment initiation (p=0. 038). In FL, treatment was associated with a trend toward higher risk of VTE, although not statistically significant (p=0. 056).

Conclusions:

DLBCL is more “thrombogenic” than FL both before and during treatment. To our knowledge, this is the first report of direct comparison of VTE event rates between different histological subtypes of NHL, knowledge of which could influence decisions related to thromboprophylaxis. There are limitations of ICD-9 codes which should be acknowledged, and further research will be required to identify the optimal method to identify patients with VTE. The lower incidence of VTE after treatment initiation in DLBCL patients suggests that the reduced disease burden associated with treatment may actually decrease the overall risk of VTE despite the increased risk associated with chemotherapy. Conversely, in FL the risk of VTE appears to be higher in association with treatment.

Disclosures:

No relevant conflicts of interest to declare.
Immunohistochemical Expression of TCRβ Predicts Achievment of Complete Remission in Enteropathy-Associated T-Cell Lymphoma
Authors: Czapiewski Piotr , Fisch Paul , Marks Reinhard , Finke Juergen , Schmitt-Graff Annette
Published: October 2019

Abstract 5101

Introduction:

Enteropathy associated T-cell Lymphoma(EATL) is a distinctive clinicopathological entity but knowledge about potential prognostic factors in this disease is very limited.

Aim:

Clinicopathological and immunohistochemical analysis of single center EATL cohort focusing especially on stromal components, activation molecules and potential loss of TCR receptors.

Methods:

Detailed clinical analysis of a cohort of 21 patients from the southern-west Germany was performed. Additionally broad panel of immunohistochemical staining including MUM1IRF4, Blimp-1, FoxP3, TCR γ, TCRβ, CD11c was done. Univariate statistical analysis of survival was performed.

Results:

The study group comprised of 12 males and 9 females, aged 37–86, with the median survival time of 5 months after the initial diagnosis. Following clinical factors turned out to be connected with longer overall survival (OS): age ≤ 60years (p=0, 007), Ann Arbor Stadium I+II (p =0, 048), good general condition (p = 0, 0068), IPI 1–3 (p=0, 004), usage of anthracycline in the chemotherapy regimen (p=0, 0504).

Longer event free survival(EFS) was connected with good general condition (p=0, 023) usage of anthracycline (p=0, 047).

There was 17 Type I cases (81%) and 4 Type II (19%). EATL type, histological variants of neoplastic cells, perivascular and angiocentric growth pattern as well as tissue eosinophilia did not influence survival. There was a strikingly strong infiltration of the neoplastic infiltrate by CD11c+ dendritic cells in 9/21 cases, but it was prognostically irrelevant. MUM1/IRF4 expression was observed in 8/19(42, 1%), Blimp-1 in 5/14 (35, 7%) and FoxP3 in 7/20 (35%), but only FoxP3 positivity was connected with longer EFS. There was a trend toward worse OS (p=0, 18) and EFS (p=0, 12) in Blimp-1 positive cases.

Expression of TCRβ was observed in 4/21(19%), of TCRγ in 3/21(14, 3%) while the majority of cases (14/21), showed loss of expression of both proteins. There was a trend toward longer OS (p=0, 19) and EFS (p=0, 16) in TCR β+ than in TCRγ+ and TCR γ-β- cases.

TCRβ expression was statistically significant more frequent in patients that achieved complete remission (3/7) than in group that did not achieve CR (1/14, p=0, 049).

Conclusions:

In EATL several clinical factors can provide relevant prognostic information. Immunohistochemical expression of FoxP3, Blimp-1 and TCRβ seems also to be prognostically usefull. TCRβ is the only marker that can predict response to the chemotherapy in this very aggressive neoplasm.

Disclosures:

Finke: Fresenius Biotech GmbH: Honoraria, Research Funding.
Detection of SNPs of MTHFR Genes in Five Hematological Malignant Tumor Cell Lines
Authors: Zhang Xiaoping , Jian Ziying , Chen Baoan , Xu Peipei , Peng Miaoxin , Xia Guohua , Cheng Lu , Lu Zuhong
Published: October 2019

Abstract 5130

Background & Aim:

This study detected three single nucleotide polymorphisms (SNPs) of methylenetetrahydrofolate reductase (MTHFR) gene in five hematology malignant cell lines, to find correlations between MTHFR gene SNPs and hematological malignancies by horizontal comparison.

Materials & Methods:

Cell line K562, Ka, HL-60, U937, and Raji were cultured. The genomic DNA was isolated by QIAamp DNA Blood Mini kit. Designing primes were amplified by PCR to get the related DNA fragments. MTHFR gene A1298C (rs1801131), C677T (rs1801131), G1793A (rs2274976) was genotyped by means of matrix assisted laser desorption ionisation-time of flight mass spectrometry method (MALDI-TOFMS).

Results:

The results showed that the genotype of locus A1298C (rs1801131) on MTHFR gene was AA for U937 cell line. While for K562, Ka, HL-60, and Raji cell lines, that was CA. For all the five hematology malignancies cell lines, the genotype of locus C677T (rs1801133) on the MTHFR gene was CC, and that of locus G1793A (rs2274976) was GG.

Conclusion:

The genotype of locus A1298C (rs1801131) on MTHFR gene was not all the same in the detected hematology malignant cell lines, while the genotypes of the other 2 loci were same in the 5 cell lines.

Disclosures:

No relevant conflicts of interest to declare.
Rituximab Is an Effective Therapy for Relapsed Thrombotic Thrombocytopenia Purpura
Authors: Jajeh Ahmad
Published: October 2019

Abstract 5139

Rituximab, a monoclonal antibodies designed against a CD20 glycoprotein expressing lymphocytes. This drug has been implemented in the treatment of B Cell Lymphoproliferative disorders as well as other non-neoplastic diseases such as autoimmune disorders. Rituximab has produced response in auto-immune thrombocytopenia, auto-immune hemolytic anemia and refractory inhibitors to coagulation factors. The proposed mechanism is not yet well known but it is postulated that rituximab affect the balance between the cytotoxic T cells and B cells. This abstract present our experience with rituximab in the treatment of relapsed Thrombotic Thrombocytopenia Purpura and Hemolytic Uremic Syndrome TTP/HUS. a total of nine patients diagnosed with relapsed TTP/HUS with mean age of forty five years; range 27– 59. Eight black females and one white male with median time to relapse of 3 months (range from 3 weeks to two years). All patients were treated initialy with plasmapheresis and steroids and achieved complete response. All maintained on tapering course of steroids for ten weeks. Upon relapse patient were given rituximab 375 mg/M2 weekly; range 3–6 weeks. This was given after treatment with plasmaphersis/steroids; range 9–25 sessions. Peripheral blood smears, LDH, CBC, BUN and creatine were monitored. In most patients schistocytes were reduced to three per 5000 red cells.

In conclusion: Rituximab is an effective therapy in relapsedTTP/HUS, larger study needed to confirm efficacy. Rituximab to be considered in the first line therapy as well as maintenance is a valid point.

Disclosures:

No relevant conflicts of interest to declare.
RHD*Weak D Type 38: A Family Study
Authors: de Menezes Costa Sidneia Sanches , Chiba Akemi Kuroda , Martin Fabio Oliveira , Langhi Dante Mario , Chiattone Carlos , Bordin Jose Orlando
Published: October 2019

Abstract 5155

Introduction:

The RHD*weak D type 38 is produced by the single nucleotide change (833G>A) in RHD exon 6, carrying a Gly278Asp substitution in the transmembraneous RhD protein. This RHD allele shows a reduced expression of the D antigen on the red blood cell (RBC) surface and may be erroneously typed as D− by standard serologic methods, including the indirect antiglobulin test (IAT). The frequency of the RHD*weak D type 38 in Caucasians is reported to be 1. 5% in Ccee phenotype, while we have recently detected a relatively higher frequency of 2. 6% among Ccee phenotype in Brazil. In this study we performed a serologic and molecular analysis of three generations from a family with the rare RHD*weak D type 38.

Study design and methods:

The RBCs from 45 years-old woman blood donor tested D negative by routine serologic tests (Ccee phenotype) but were positive by the adsorption-elution technique. Further serologic investigation to establish the patterns of reactivity of the RBCs was preformed using anti-D Moabs IgM (clones HM10, P3X63, P3X21211F1, P3X21223B10; Diagast, Loos, France) and anti-D Moabs IgG/IAT (clones HM16, P3X35, P3X241, P3X249, P3X290; Diagast, Loos, France, and MS-26, ESD1; DiaMed, Latino América S. A.). Flow cytometric methods were employed to evaluate the D antigen densities, and molecular sequencing studies were done using a sequencing Kit (Big Dye Terminator v1. 1, applied Biosystems, Weiterstadt, Germany) and a genetic analyser (ABI 3100, Applied Biosystems, Foster City, CA, USA).

Results:

We found that the RBCs reacted negatively with 4/4 IgM anti-D, but showed weakly positive results with 4/7 IgG anti-D reagents. In face of such results we studied 12 members of her family and found that 6 more individuals tested D negative by standard serologic tests (Ccee phenotype) but were positive by adsorption-elution technique. The flow cytometry investigation of samples from the 7 subjects showed D antigen density in a range of 60 – 80 sites per cell and a Rhesus Index of 0. 1 – 0. 6. DNA sequencing analysis from 7 family members was performed showing that all descendants (siblings) had the same RHD*weak D type 38 genotype as the mother, also inherited by the grandson.

Conclusions:

The molecular analysis identified the presence of RHD*weak D type 38 in the three generations of a Brazilian family. The study revealed maternal inheritance (first generation) of the weak D type 38, with Ccee phenotype by her six children and the second generation showed paternal inheritance in a son. We also emphasize the significant prevalence of RHD*weak D type 38 in the Brazilian population, often showing a low antigen D density.

Disclosures:

No relevant conflicts of interest to declare.
Study of Renal Iron Overload by T2* MRI in a Large Cohort of Thalassemia Major Patients
Authors: Meloni Antonella , De Marchi Daniele , Positano Vincenzo , Giuffrida Gaetano , Armari Sabrina , Campisi Saveria , Furbetta Mario , Maggio Aurelio , Valeri Gianluca , Chiodi Elisabetta , Lombardi Massimo , Pepe Alessia
Published: October 2019

Abstract 5177

Background.

Renal dysfunction has been reported in adult subjects with thalassemia major (TM) since 1975. One of the main cause is the iron overload consequent to regular transfusions. Multiecho T2* MRI is a well-established technique for cardiac and hepatic iron overload assessment, but there very few report concerning the kidneys.

The aims of this study were to describe the T2* values of the kidneys in patients with TM, to investigate the correlation between renal and myocardial or hepatic siderosis and biventricular cardiac function.

Methods.

119 TM patients (58 men, 30. 7 ± 8. 2 years) enrolled in the Myocardial Iron Overload (MIOT) networks underwent MRI. For the measurement of iron overload, multiecho T2* sequences were used. The left ventricle was segmented into a 16-segments standardized model and the T2* value on each segment was calculated as well as the global value. In the liver, the T2* value was assessed in a single region of interest (ROI) in a homogeneous area of the parenchyma. For each kidney, T2* values were calculated in three different ROIs and were averaged to obtain a representative value for the kidney. The mean T2* value over the kidneys was also calculated. Cine images were obtained to quantify biventricular morphological and functional parameters in a standard way.

Results.

T2* values in the right kidney were significant lower than in the left kidney (40. 3±11. 9 ms vs 44. 1±12. 7 ms, P<0. 0001). The mean T2* value over the kidneys was 42. 2±11. 9 ms and 40 patients (33. 6%) had a pathological value (T2*<36 ms, lower limit of normal evaluated on 20 healthy subjects). The mean T2* value did not show a significant difference amongst men ad women (43. 2±11. 7 ms versus 41. 3±12. 1 ms, P=0. 378). The mean T2* values increased with age in a significant manner (r=0. 321, P<0. 0001). There was a significant negative correlation between serum ferritin levels and mean renal T2* values (r=-0. 446, P<0. 0001). Significant positive correlations of the mean T2* values were demonstrated for liver (r=0. 511, P<0. 0001) and global heart (r=0. 262, P=0. 004) T2* values (Figure 1). No correlation was found between renal iron overload and bi-ventricular function parameters.

Conclusions.

Systemic T2* differences between left and right kidneys were found, with significant lower values in the right one. Mean T2* value increased with age. We confirmed that kidney iron deposition was not very common in TM, but it was correlated with iron deposition in liver and heart.

Disclosures:

No relevant conflicts of interest to declare.
Genetic Heterogeneity of Alpha - and Beta - Globin Mutations in a German Multiethnic Population Group
Authors: Grosse Regine , Oyen Florian , Weber Belinda , Schneppenheim Reinhard
Published: October 2019

Abstract 5185

Thalassemia syndromes are among the most common hereditary diseases worldwide and are widespread throughout the Mediterranean Region, Africa, the Middle East, India, Burma, the Southeast Asia and Indonesia. Similarly, hemoglobinopathies are common in different ethnic groups and mixed syndromes of Thalassemia and hemoglobinopathies exist.

In this retrospective study we report the number and type of Thalassemia mutations and their combination with hemoglobinopathies detected mostly in individuals with a migration background now living in Germany.

DNA samples were analyzed by polymerase chain reaction (PCR) and direct DNA-sequencing and additionally by multiplex ligation-dependent amplification (MRC-HOLLAND MLPA®).

Mutations in one or both beta-globin chains were found in 77 patients. Most of these mutations were beta Thalassemia mutations, n=41 (Thalassemia minor n=33, Thalassemia major/intermedia n=7, homozygous Quin-Hai Hemoglobinopathy n=1).

In 34 samples we found the hemoglobin S single base mutation c. 20A>T p. E7V, either alone or in combination with a hemoglobin C mutation c. 19G>A p. E7K or beta Thalassemia mutation (HbAS n=14, HbSS n=12, HbSC n=6, HbSThal n=2).

In two patients we found only the hemoglobin C mutation by itself (HbAC n=1, HbCC n=1).

In 52 samples we found mutations on the alpha-globin chains, most often the 3. 7KB deletion (n=27), followed by the SEA (n=4), the 20. 5 KB deletion (n=4), the 4. 2 KB deletion (n=4), the Dutch deletion (n=3) and alpha triplication (n=3). Two of the remaining seven samples showed new alpha Thalassemia mutations which have not been described yet.

In an additional 34 DNA samples we found a combination of alpha- and beta-globin chain changes. In 4 of these samples we detected new mutations in the alpha-globin chains.

The prevalence of mutations in the alpha- and beta-globin chains varies greatly because of a complex ethnic structure of our patients. Changes of the alpha-globin chains could not be safely detected by hemoglobin-electrophoresis. These situations are very important for genetic counseling in a population in which consanguineous marriages are common. Furthermore, alpha-globin mutations are genetic modifiers for beta Thalassemia and sickle cell disease and will influence the phenotype of beta-globin chain mutations. Patients with a compound heterozygous mutation for beta Thalassemia will probably not be detected by hemoglobin-electrophoresis alone, as in milder forms and early childhood the hemoglobin is not severely decreased and hemoglobin electrophoresis may be misinterpreted for Thalassemia minor.

Our findings underline the heterogeneity of beta-globin and alpha-globin chain mutations and the importance of hematological and molecular analyses in the diagnosis and genetic counseling.

Disclosures:

No relevant conflicts of interest to declare.
Adenoviral E4 Gene Stimulates Secretion of PEDF That Maintains Long-Term Survival of Human Glomerulus-Derived Endothelial Cells
Authors: Nekhai Sergei , Kumari Namita , Obuhkov Yuri , Jerebtsova Marina
Published: October 2019

Abstract 5187

Renal glomerular endothelial cells are specialized cells with an important role in physiological filtration and glomerular disease. However, maintenance of human primary endothelial cells requires stimulation with serum and growth factors that often results in modification of the cells properties. Previously, expression of early adenovirus region E4 was shown to help maintaining long-term survival of human endothelial cells in serum free media without addition of growth factors. In the current study, we showed that media conditioned with human epithelial cells stably transfected with Ad E4 region supported survival of human glomerulus-derived endothelial cells in serum-free media (Fig. 1 A and B). Mass-spectrometry analysis of the conditioned media identified pigmental epithelium derived factor (PEDF) as a major component of the conditioned media. PEDF expression in 293-E4 cells was validated by RT- PCR (Fig. 1C), Western Blot and ELISA analysis (Fig. 1D). PEDF expression was detected in mouse glomeruli. Supplementation with recombinant PEDF supported survival of primary endothelial cells and the cells transformed with SV40 large T antigen in serum-free media, and extended the life-span of both cell cultures. PEDF did not inhibit FGF-2 stimulated growth and tubulogenesis of endothelial cells. Thus we demonstrated that adenoviral E4 region stimulated expression and secretion of PEDF by human renal epithelial cells that acted as a survival factor for glomerulus-derived endothelial cells.

Acknowledgments:

This work was supported NIH Research Grants SC1GM082325, R25 HL003679, 2G12RR003048, 8G12MD007597, K25GM097501 and 1P30HL107253. This study was also supported in part by National Kidney Foundation grant.

Disclosures:

No relevant conflicts of interest to declare.
BCR-ABL1 Kinase Inhibits DNA Glycosylases to Enhance Oxidative DNA Damage and Stimulate Genomic Instability
Authors: Slupianek Artur , Falinski Rafal , Znojek Pawel , Stoklosa Tomasz , Flis Sylwia , Doneddu Valentina , Synowiec Ewelina , Blasiak Janusz , Bellacosa Alfonso , Skorski Tomasz
Published: October 2019

Abstract 520

Tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib revolutionized the treatment of BCR-ABL1 kinase-positive chronic myeloid leukemia in chronic phase (CML-CP). Unfortunately, 15–25% of patients initially responding favorably to imatinib will develop acquired drug resistance, which in 40–90% of cases is caused by genomic instability resulting in the appearance of clones expressing TKI resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species (ROS) resulting in excessive oxidative DNA damage such as oxidized DNA bases (8-oxoguanine and 5-hydroxycytosine→uracil) (Nieborowska-Skorska et al., Blood, 2012). Unfaithful and/or inefficient repair of these lesions generates TKI resistant point mutations in BCR-ABL1 kinase. Oxidative DNA lesions may be removed by base excision repair (BER) or, if not removed, will create mismatches, which are repaired by mismatch repair (MMR). Since we found that MMR is inhibited in CML-CP (Stoklosa et al., Cancer Res., 2008), the activity of BER is critical to prevent the accumulation of point mutations. Using an array of specific substrates and inhibitors/blocking antibodies we found that two major glycosylases, uracil-DNA glycosylase UNG2 and 8-oxoguanine glycosylase (OGG1) responsible for the excision of uracil (product of oxidation of cytosine) and 8-oxoguanine (8-oxoG) from DNA, respectively, were inhibited in BCR-ABL1 –transformed cell lines and CD34+ CML cells. The inhibitory effect was even more pronounced in CML blast phase (CML-BP) in comparison to CML-CP, it depended on BCR-ABL1 kinase activity and was not accompanied by deregulation of nuclear expression and/or chromatin association of these glycosylases. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases such as TEL-ABL1, TEL-PDGFbetaR and NPM-ALK did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI we found that UNG2 activity diminished the number of oxidized DNA bases detected by modified comet assay and prevented accumulation of point mutations in reporter gene Na+/K+ATPase, which encode resistance to ouabain. In conclusion, we hypothesize that inhibition of UNG2 and OGG1, accompanied by reduced MMR activity is responsible for accumulation of TKI-resistant BCR-ABL1 kinase point mutations and perhaps also other point mutations facilitating malignant progression of CML.

Disclosures:

No relevant conflicts of interest to declare.
Refinement in Patient Selection to Reduce Treatment-Related Mortality From Stem Cell Transplantation in Amyloidosis
Authors: Gertz Morie A , Lacy Martha Q , Dispenzieri Angela , Kumar Shaji K. , Dingli David , Leung Nelson , Hogan William J , Buadi Francis K , Hayman Suzanne R
Published: October 2019

Abstract 599

Purpose:

This study was undertaken to develop selection guidelines for transplant centers to determine eligibility for stem cell transplantation in patients with light chain amyloidosis.

Patients and Methods:

Patients with biopsy-confirmed immunoglobulin light chain amyloidosis who underwent stem cell transplantation between March 8, 1996, and December 31, 2011, were reviewed in 2 cohorts: those who underwent transplantation between March 8, 1996, and June 30, 2009, and those who underwent transplantation between July 1, 2009, and December 31, 2011 (table 1). A second comparison was undertaken among patients who died before posttransplant day 100 to determine features predictive of early death (table 2).

Results:

A total of 499 patients were identified, 410 in the earlier group and 89 in the later group. After July 1, 2009, significantly fewer transplant recipients had Mayo stage III cardiac involvement. Mortality before posttransplant day 100 was 10.5% (43/410) in the earlier group and 1.1% (1/89) in the later group. In the earlier group, one-quarter of transplant recipients with N-terminal pro-brain natriuretic peptide (NT-proBNP) levels higher than 5,000 pg/mL died by 10.3 months (fig 1, left). When the serum troponin T level was > 0.06 ng/mL, 25% died at 3.7 months (fig 2, right).

Conclusion:

The Mayo staging system is highly predictive for overall survival but not useful for selecting transplant recipients. Patients with serum troponin T levels higher than 0.06 ng/mL or NT-proBNP levels >5,000 pg/mL (not on dialysis) should not be considered acceptable candidates for stem cell transplantation because of unacceptable early mortality.

Disclosures:

No relevant conflicts of interest to declare.
MLL-Rearranged Leukemias Are Dependent On the Histone H2B Ubiquitin Ligase RNF20
Authors: Vakoc Christopher R. , Wang Eric , Kawaoka Shinpei
Published: October 2019

Abstract 649

Histone mono-ubiquitination is a major signaling mechanism that shapes the structure and function of chromatin. Hence, factors that regulate ubiquitin attachment to histones (E2 conjugating enzymes, E3 ligases, and de-ubiquitinating enzymes) often play fundamental roles in epigenetic biological processes, including the pathophysiology of human disease. Here, we explored whether the known regulators of histone ubiquitination/deubiquitination are linked to the pathogenesis of acute myeloid leukemia, a disease often associated with aberrant chromatin signaling. To this end, an shRNA screen was performed where all histone E2s, E3s, and deubiqitinating enzymes were individually suppressed and evaluated for their role in supporting growth of acute myeloid leukemias initiated the MLL-AF9 oncogene. This analysis identified the histone H2B ubiquitin ligase RNF20 (also known as BRE1A) as essential for MLL-AF9 leukemia growth. This finding was verified in a genetically engineered mouse leukemia model (driven by MLL-AF9 together with NrasG12D) under both in vitro and in vivo conditions. In addition, human leukemia cell lines harboring MLL-rearrangements were found to be uniformly dependent on RNF20 for their proliferation. Upon inhibition of RNF20 and elimination of global H2B mono-ubiquitination, leukemia cells arrest in the G1/G0 stage of the cell-cycle without evidence of myeloid maturation. RNA-SEQ analysis identified that a suite of direct transcriptional targets of MLL-AF9 were downregulated in expression upon RNF20 inhibition, including Hoxa9, Hoxa10, Meis1, and Mef2c. While H2B mono-ubiquitination has been shown previously to promote the catalytic function of H3K4 and H3K79 methyltransferases, we find that in leukemia cells inhibition of RNF20 eradicates global H2B mono-ubiquitination without influencing global H3K4me3 and H3K79me2/me3. However, we find instead that RNF20 is selectively required to maintain local levels of H3K79 methylation in the body of MLL-AF9 target genes, such as Hoxa9. Since MLL-AF9 is known to directly recruit the H3K79 methyltransferase DOT1L to transactivate its target genes, our findings suggest a mechanism whereby targeting RNF20 interferes with the transactivation function of MLL-AF9. Moreover, we found that shRNA-based suppression of RNF20 sensitizes leukemia cells to the growth-arrest induced by the BET bromodomain inhibitor JQ1, without influencing the sensitivity to cytotoxic agents daunorubicin and etoposide. Based on these collective findings, we propose that RNF20 represents a major chromatin-based signaling pathway necessary to maintain the MLL-rearranged subtype of acute myeloid leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Carfilzomib, Cyclophosphamide and Dexamethasone (CCd) for Newly Diagnosed Multiple Myeloma (MM) Patients
Authors: Palumbo Antonio , Bringhen Sara , Villani Oreste , Siniscalchi Agostina , Russo Eleonora , Uccello Giuseppina , Cerrato Chiara , Gilestro Milena , Rossi Davide , Boccadoro Mario
Published: October 2019

Abstract 730

Background:

Carfilzomib is a novel, irreversible proteasome-inhibitor with a significant activity and favourable toxicity profile. Here we evaluated efficacy and safety of the combination carfilzomib-cyclophosphamide-dexamethasone in elderly (≥ 65 years) newly diagnosed MM patients.

Methods:

The Bryant and Day two-stage design was used to evaluate both efficacy and safety. Patients received oral cyclophosphamide (300 mg/m2 on days 1,8,15), oral dexamethasone (40 mg on days 1, 8, 15, 22) and iv carfilzomib (20 mg/m2 on days 1, 2, and 36 mg/m2 on days 8, 9, 15, 16, cycle 1; 36 mg/m2 on days 1, 2, 8, 9, 15, 16, cycles 2–9) every 28 days for 9 cycles, followed by maintenance with iv carfilzomib (36 mg/m2 on days 1, 2, 15, 16) every 28 days until progression or intolerance.

Results:

Thirty-four patients were enrolled: median age was 70 years, 21% of patients were older than 75 years, 24% had reduced creatinine clearance (<50 mL/min), 46% had ISS stage III, and 28% had unfavorable FISH [t(4;14) or t (14;16) or del17p]. Nineteen patients were evaluated for response and for safety after at least 4 cycles (table). All patients achieved at least partial response (PR), 74% at least very good partial response (VGPR), 42% at least complete response (CR)/ near-CR, including 10% stringent-CR. Responses were rapid with the median time to PR of 1 month and the median time to CR of 2 months. After a median follow-up of 7.5 months no patient has progressed or died.

Grade (G) 4 hematologic toxicities included neutropenia (1 patient, 5%). At least one G3-4 non-hematologic event was reported in 4 patients (21%): pneumonia and bronchitis (2 patients, 10%), atrial fibrillation (1 patient, 5%) and renal failure (1 patient, 5%). Only 1 patient (5%) developed G1 peripheral neuropathy. No patient discontinued treatment and 4 patients (21%) required carfilzomib dose reductions due to adverse events (G4 neutropenia, G3 pneumonia, G3 atrial fibrillation and G3 renal failure).

Conclusions:

Carfilzomib-cyclophosphamide-dexamethasone showed encouraging activity in patients with newly diagnosed MM. The combination was well tolerated with a frequency of at least one G3-4 non-hematologic adverse event of 21%. Results will be updated at the meeting.

Disclosures:

Palumbo: Onyx: Honoraria. Bringhen:Onyx: Honoraria.
A Calmodulin Interacting Protein Iqcg Is Required for Definitive Hematopoiesis in Zebrafish
Authors: Chen Li-Ting , Liang Wen-Xue , Chen Shuo , Chen Zhu , Chen Sai-Juan
Published: October 2019

Abstract 764

We previously identified a novel fusion gene NUP98-IQCG in a bi-phenotypic acute T-lymphoid/myeloid leukemia patient. However, the function of IQCG has never been demonstrated. By taking advantage of the zebrafish as a model organism, we investigated the role of IQCG in hematopoietic development and found that the definitive hematopoiesis was severely impaired in the iqcg deficient embryos. The hematopoietic stem cell (HSC) population as well as the number of multilineage differentiated hematopoietic cells was reduced. Moreover, the number of phosphorelated histone3 positive HSC was decreased, which indicated a compromised proliferation in iqcg deficient HSC.

Since IQCG protein contains an IQ calmodulin binding motif (IQ motif), we next examined if IQCG protein can interact with calmodulin. Co-immunoprecipitation assay results showed that both human and zebrafish IQCG proteins have the ability to precipitate calmodulin. Deletion of IQ motif disrupted this interaction. The isothermal titration calorimetry results indicated that the IQ motif of IQCG protein have a lower association constant with calcium-loaded calmodulin than apo-calmodulin. We next crystallized the complex of calmodulin and IQ motif of IQCG in two circumstances-with and without calcium. Determination of these two crystal structures indicated different interaction details between them. These results suggest that IQCG may be involved in calcium signaling in HSC.

Previous studies reported that calmodulin dependent protein kinase IV (CaMKIV) involves in HSC maintenance and survival. So we wondered if CaMKIV acts downstream of IQCG. Indeed, whole-mount immunofluorescence data showed that the level of phosphorelated CaMKIV markedly reduced in iqcg morphants compared with controls. Reactivation of CaMKIV by co-injection with constitutively activated CaMKIV (CACaMKIV) mRNA rescued the HSC number in iqcg deficient embryos. Furthermore, embryos injected with camkIV morpholino phenocopied the iqcg morphants.

In summary, we demonstrate an important role of the novel NUP98 partner gene IQCG in definitive hematopoietic development. IQCG may control the HSC proliferation through interacting with calmodulin and acting upstream of CaMKIV, which suggests a novel pathway in definitive hematopoietic development.

Disclosures:

No relevant conflicts of interest to declare.
RB Controls Size, Cellularity, and T Cell Output of the Mouse Thymus
Authors: Garfin Phillip M. , Viatour Patrick , Min Dullei , Bryson Jerrod , Weinberg Kenneth I. , Manley Nancy R. , Sage Julien
Published: October 2019

Abstract 835

The establishment of the thymic microenvironment early in life is crucial for the production functional T cells. Conversely, thymic involution results in a decreased T cell output. Thymic involution has important health implications especially following bone marrow transplant. Our objective is to determine molecular and cellular mechanisms that will allow for regeneration of involuted thymic tissue, restore production of naïve T cells, and improve immune function while improving our understanding of immunobiology. In this pursuit, we have focused on the Retinoblastoma family of tumor suppressor proteins. The main function of the RB pathway is to restrict passage through the G1/S transition of the cell cycle. RB and its two family members, p107 and p130, mediate the action of a broad range of cellular signals to control the proliferation, survival, and differentiation status of a large number of mammalian cell types. We found that inactivation of the RB pathway in the thymus by early deletion of RB family genes prevents thymic involution, promotes expansion of functional thymic epithelial cells (TECs), and increases thymic T cell output. Moreover, we have identified a direct regulatory relationship between RB and the Foxn1 transcription factor Via E2F transcription factors, where RB/E2F complexes directly repress the Foxn1 promoter, thereby promoting involution. Thus, the RB family is a critical mediator of extra- and intra-cellular signals to regulate thymic epithelial cells and thymus function, and decreasing RB pathway function may promote regeneration of the involuted thymus and restoration of naïve T cell production in patients.

Disclosures:

No relevant conflicts of interest to declare.
Histone Deacetylase 11 (HDAC11) Regulates Cytotoxic T-Cell Function and Memory Phenotype
Authors: Woods David M , Woan Karrune V. , Sahakian Eva , Powers John , Cheng Fengdong , Wang Hongwei , Rock-Klotz Jennifer , Weber Jeffrey S. , Villagra Alejandro , Pinilla-Ibarz Javier , Sotomayor Eduardo M.
Published: October 2019

Abstract 840

T-cells are an essential component of immune mediated tumor rejection. Adoptive transfer of T-cells results in a durable anti-tumor response in some patients with hematological malignancies. To further improve the efficacy of T-cell adoptive transfers, a better understanding of the regulatory checkpoints of these cells is needed. Here we show that HDAC11 is a negative regulator of CD8+ T-cell function, thus representing a potential target in adoptive immunotherapy. HDACs are a group of enzymes initially known for their role in deacetylating histones, thereby condensing chromatin structure and repressing gene expression. The known roles of HDACs as epigenetic regulators have recently expanded to include more complex regulatory functions including interactions with non-histone targets. HDAC11 is the most recently identified member of the HDAC family, and is highly expressed in brain, testis and T-cells. Recently, our group reported HDAC11 as a regulator of IL-10 production in antigen presenting cells. To determine the role of HDAC11 in T-cell biology, T-cells from HDAC11 knock out (HDAC11KO) mice were compared to wild-type T-cells in number, function and phenotype. HDAC11KO T-cells had no differences in absolute number or percentages of CD4+ or CD8+ lymphocytes. However CD8+ T-cells were hyper-proliferative upon CD3/CD28 stimulation and produced significantly higher levels of the pro-inflammatory, Tc1 cytokines IL-2, INF-γ, and TNF-α. However, no significant increases in the production of the Tc2 cytokines IL-4, IL-6 or IL-10 were seen. Further investigation of phenotypic differences also revealed that HDAC11KO mice have a larger percentage of central memory CD8+ T-cells. Additionally, HDAC11KO CD8+ T-cells express higher levels of the transcription factor Eomes, a known contributor to central memory cell formation as well as a controller of granzyme B and perforin production in CD8+ T-cells. This Tc1 and central memory-like phenotype translated to delayed tumor progression and survival in vivo in C1498 AML bearing mice treated with adoptively transferred HDAC11KO T-cells, as compared with wild type T-cells. Collectively, we have demonstrated HDAC11 as a negative regulator of CD8+ T-cell function, and a novel potential target to augment the efficacy of adoptive T-cell tumor immunotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Rasgrf1 Overexpression Amplifies B Cell Receptor Signaling in Chronic Lymphocytic Leukemia Specimens
Authors: Liao Wei , Jordaan Gwen , Sharma Sanjai
Published: October 2019

Abstract 926

The B-cell receptor (BCR) signaling is a hallmark of chronic lymphocytic leukemia cells and results in cell proliferation, survival and resistance to apoptosis. B cell receptor (BCR) signaling in CLL specimens activates the Ras/ERK/MAPK pathway and is critical for survival and growth of CLL cells. Rasgrf1 is a guanine exchange factor that accelerates the removal of inactive GDP from Ras so that active GTP can bind Ras that results in Ras activation. In an Affymetrix microarray expression analysis screen of CLL specimens (n=5) and peripheral blood normal B cells (n=2), Rasgrf1 was found to be over-expressed (ranked within top 100 overexpressed genes) in CLL. This finding was further confirmed by quantitative RT-PCR as a 5–300 fold overexpression in CLL specimens (n=20) was noted as compared to normal B cells. Western blot analysis data also showed over-expression of Rasgrf1 protein in CLL specimen lysates but interestingly the expressed protein is of a smaller size in CLL specimens (70-80 kDa) as compared to the Rasgrf1 in normal peripheral blood mononuclear cells (140 kDa) This truncated protein retains the important c-terminal GEF (guanine exchange factor) domain but lacks the regulatory domains and is known to function as a hyperactive GEF in other tumor model systems. Knockdown of Rasgrf1 in CLL specimens (n=4) resulted in inhibition of the BCR-Ras/ERK/MAPK pathway as determined by a decrease in ERK phosphorylation. Similar Rasgrf1 knockdown experiments with BCR crosslinking also showed identical results in additional CLL specimens. Transient SiRNA mediated Rasgrf1 knockdown also resulted in higher percentage of annexin positive or apoptotic CLL cells as compared to the scrambled SiRNA control transfection. Lysates from the Rasgrf1 knockdown cells showed an increase in PARP cleavage by western blot analysis as well indicating that continued expression of this protein was necessary for survival of the leukemic cells. To confirm that the Rasgrf1 knockdown resulted in a decrease in the GEF function, the active Ras (Ras-GTP) in the CLL specimens was evaluated by an ELISA assay. In two CLL specimens the assay confirmed that the active Ras in the CLL cells was decreased with Rasgrf1 knockdown. Overall the findings implicate a novel GEF protein, Rasgrf1 as an amplifier of BCR signaling in CLL cells.

Disclosures:

No relevant conflicts of interest to declare.
Erythropoietin-Directed Erythropoiesis Depends Upon Serpin Inhibition of Erythroblast Lysosomal Cathepsins
Authors: Wojchowski Don M. , Dev Arvind , Byrne Susan M , Verma Rakesh , Ashton-Rickardt Philip G
Published: October 2019

Abstract 981

EPO is used to treat anemia associated with chronic kidney disease, and cancer chemotherapy. However, EPO has hypertensive and thrombolytic side-effects and can impact on innate immunity, diabetes, vasculogenesis, and the progression of certain cancers. These observations, together with the clinical emergence of new EPOR agonists provide compelling reasons to better understand key EPO/EPOR actions. This includes effects on erythroid progenitor cells (EPC's) as a prime target in which EPO/EPOR actions remain incompletely understood. To discover novel EPO action modes we profiled the transcriptome of primary EPC's. We report Serpina3g/Spi2A as a major EPO/EPOR target, and a key EPC survival factor. In knockout mice, loss of Spi2A sharply worsened anemia due to hemolysis, radiation or transplantation (p = 0.008; p = 0.0001; p = 0.010, respectively). EPO-induced red cell formation also was limited to 8.6 + 0.7% of wild-type control responses. Unexpectedly, maturing erythroblasts proved to require Spi2A for cytoprotection, with iron and ROS defined as cytotoxic agents. Notably, Spi2A defects were ameliorated by cathepsin-B/L inhibition, and by the genetic co-deletion of lysosomal cathepsin-B. In vivo, pharmacological inhibition of cathepsin B/L enhanced EPO's effects on hematocrits by 5+ points (d4 and d6 post- EPO). Overall, we define an unexpected EPO action mode via an EPOR- Spi2A serpin- cathepsin axis in maturing erythroblasts, with oxidants (and iron) as cytotoxic potentiators, and lysosomal cathepsins as novel therapeutic targets.

Disclosures:

No relevant conflicts of interest to declare.
Divalent Metal Transporter 1 (DMT1) Regulates EPO Receptor Gene Expression Via GATA-1
Authors: Burda Pavel , Curik Nikola , Horvathova Monika , Divoky Vladimir , Stopka Tomas
Published: October 2019

Abstract 991

Introduction:

Erythroid differentiation and iron metabolism are interconnected processes in order to produce sufficient numbers of appropriately hemoglobinized red cells. Patients carrying mutations of divalent metal transporter DMT1 display severe microcytic anemia and iron overload. In vivo, this defect can be partially rescued by stimulation of erythropoiesis by erythropoetin (EPO). In vitro, addition of EPO together with iron-saturated salicylaldehyde isonicotinoyl hydrazone (Fe-SIH, a non-transferrin iron donor) to the cultures significantly improved the growth of patient's DMT1-mutant erythroid progenitors (Horvathova et al, 2012). Regulation of erythropoiesis by EPO and its receptor (EPOR) involves transcription factor GATA-1. Our earlier data (Burda et al, 2009) showed that in erythroblasts GATA-1 transcriptionally regulates both Epor and Dmt1 and that this activation is blocked by negative regulator of erythroid differentiation, transcription factor PU.1. This suggests that simultaneous expression of EPOR and DMT1 is required for erythroid differentiation and survival of erythroid cells. We hypothesize that deficiency of DMT1 negatively affects expression of EPOR, thus leading to inhibition of EPO/EPOR signaling. We suppose that this inhibition involves GATA-1 and PU.1.

Methods:

We used mouse erythroleukemia (MEL) cells expressing 17-OH-estradiol-inducible transgenes of GATA-1 (GER) or PU.1 (PUER). mRNA was quantitated by RT-PCR and protein occupancy on DNA was determined by chromatin immunoprecipitation (ChIP). Downregulation of Dmt1 was achieved by siRNA.

Results:

Using ChIP we established that GATA-1 and PU.1 regulate Epor gene directly. By activating GER or PUER in MEL cells we observed that promoter region of Epor is enriched and depleted respectively by acetylated histone H3 lysine (K) 9. Furthermore, inhibition of Epor by PU.1 coincided with recruitment of PU.1 to the Epor promoter. Similar ChIP analyses of Dmt1 promoter are undergoing. We next activated GER in MEL cells in the presence or absence of Dmt1 siRNA and observed that Dmt1 expression is needed for GATA-1-dependent upregulation of Epor mRNA expression during erythroid differentiation.

Conclusion:

We found that GATA-1 stimulates both Epor and Dmt1 expression and that PU.1 blocks directly these effects. We also found that erythroid transcriptional regulation of Epor via transcription factor GATA-1 is severely diminished in Dmt1-knockdown MEL cells. We are currently testing whether this effect involves abnormal transport of iron or other divalent metals.

(Grant support: P305/11/1745, P301/12/P380, P305/12/1033. Institutional funding: PRVOUK-P24/LF1/3, UNCE 204021, SVV-2012–264507, GAUK 251135 82210).

Disclosures:

No relevant conflicts of interest to declare.