bluuebunny/crossref_metadata_2025 huggingface dataset:
The B Cell Receptor Antagonist R406 Alters The Balance Between Anti-Apoptotic and Pro-Apoptotic Signals To Strongly Synergize With ABT-199 In Diffuse Large B Cell Lymphoma Cells
Authors: Zhang Yue , Francoise Powell , Devereaux Erik , Passino Melissa , Parmentier Julie , Byth Kate
Published: October 2019

SYK plays an essential role in B cell receptor (BCR) mediated survival in Diffuse Large B Cell Lymphoma (DLBCL). In a previous clinical investigation, fostamatinib, a potent Syk inhibitor, showed evidence of anti-tumour activity with an overall response rate of ∼20% in a cohort of DLBCL patients (JW Friedburg et al Blood. 2010, 115:2578-85). This finding prompted us to investigate the mechanism of action of fostamatinib in DLBCL. Our previous work demonstrated R406, the metabolic active form of fostamatinib, functions as a BCR antagonist in DLBCL cells. Treatment with R406 reduced MCL1 and down-regulated Bfl-1 expression, leading to apoptosis induction in sensitive cells. Due to R406’s impact on apoptosis machinery, here we explored the ability of R406 to combine with a selective Bcl2 inhibitor, ABT-199 in DLBCL cells.

Drug combinations were tested in DLBCL cells and synergy scores were determined using a 6x6 dose matrix followed by analysis via Chalice software (Zalicus). ABT-199 strongly synergized with R788, the prodrug of R406, in multiple R406 sensitive, but not resistant cell lines. In contrast, little effect was observed in combinations of R788 with other reagents, including ibrutinib (PCI-32765), bortezomib, CAL-101 and dasatinib. Flow-cytometry apoptosis analysis confirmed that R406 enhanced ABT-199-induced apoptosis in both ABC subtype and GCB subtype DLBCL cells. Further studies demonstrated R406 not only decreased anti-apoptotic proteins, but also activated pro-apoptotic proteins. Specifically, R406 suppressed ERK-dependent phosphorylation of BIM on S69, resulted in stabilization of BIM. In addition, R406 induced dephosphorylation of BAD at S136 in sensitive but not resistant cells, likely through inhibition of AKT kinase activity. Furthermore, treatment of R406 also upregulated other pro-apoptotic BH3-only proteins such as HRK1 and BMF1, which may partially contribute to the observed synergy.

In conclusion, we have demonstrated R406 strongly synergizes with ABT-199 in vitro, likely by altering the balance between anti-apoptotic and pro-apoptotic signals in DLBCL cells. This may provide a potential combination therapy to be tested in DLBCL.

Disclosures:

Zhang: AstraZeneca: Employment, Possible shareholder Other. Francoise:AstraZeneca: Employment, Possible shareholder Other. Devereaux:AstraZeneca: Employment, Possible shareholder Other. Passino:AstraZeneca: Possible shareholder Other. Parmentier:AstraZeneca: Possible shareholder Other. Byth:AstraZeneca: Employment, Possible shareholder Other.
Targeting BTK As a Treatment For Multiple Myeloma Stem Cells
Authors: Yang Ye , Gu Zhimin , Xu Hongwei , Huang Junwei , He Jie , Tao Yi , Tricot Guido J. , Zhan Fenghuang
Published: October 2019

Background

Multiple myeloma stem cells (MMSCs), characterized by drug-resistance and self-renewal, are very likely responsible for relapse. Though the phenotypic features of MMSCs are still controversial, it is believed that MMSCs are enriched in plasma cell progenitors. Bruton’s tyrosine kinase (BTK) plays a central role in B-cell development and plasma cell differentiation. BLAB/c. CBA/N mice carrying the defective BTK gene are resistant to pristine-induced plasmacytomagenesis suggesting activation of BTK is essential for plasma tumor initiation. In this study, we explore the role of BTK in maintaining MMSCs.

Materials and methods

Real-time-PCR was used to detect the BTK expression in MMSCs and compared to the bulk cells. BTK-related self-renewal and drug resistance were evaluated by clonogenic formation assays, using 10,000 MM cells from the BTK over-expressing ARP1 and OPM2 MM cell lines, treated with bortezomib (1nM, 10nM), as well as doxorubicin (50nM, 100nM), etoposide (50nM, 100nM), and verapamil (50nM). Flow cytometry was performed to detect the side population (SP) fraction and apoptosis in MM cells. Co-immunoprecipitation assay and western blot were utilized for the mechanistic BTK studies. Effect of BTK on MM progression in vivo was assessed in the 5TGM1 myeloma mice and NOD/SCID mice models.

Results

Our results revealed that BTK, iPS genes, Wnt and Hedgehog signaling pathways were significantly increased in MMSCs (CD138- and SP fraction) compared to bulk myeloma cells in both MM cell lines and primary MM cells. We also demonstrated that over-expression of BTK in the low-expressing MM cell lines, ARP1 and OPM2 increased the SP fraction, clonogenic potential and drug-resistance, while knockdown of BTK in high-expressing MM cell lines, OCI-MY5 and H929 abrogated these characteristics. Our in vivo study indicated that over-expression of BTK in ARP1 cells accelerates tumor formation in NOD/SCID mice compared to the EV control cells. Targeting BTK by shRNA or BTK inhibitor, CGI-1747 delayed MM tumor formation and MM progression in NOD/SCID mice and 5TGM1 model, respectively. Mechanistic studies identified that BTK activates the AKT pathway, inducing the drug-resistant genes, p-Bcl-2 and ABCB1, and inhibiting GSK3β leading to activation of the Wnt/ β-Catenin pathway. Inhibition of AKT by a specific inhibitor decreased Wnt and Hh proteins by western blot.

Conclusion

BTK plays an important role on maintaining MM stemness and drug-resistance through activating AKT, Wnt and Hh signaling pathways. Targeting BTK in MM may be a promising strategy to eliminate MMSCs.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Responses At 3 Months Are Associated With a Better Subsequent Molecular response and Outcome In Children and Adolescents With CML In Chronic Phase: Results Of a Retrospective Analysis From The French Glivec Phase 4 Study
Authors: Millot Frédéric , Guilhot Joelle , Baruchel André , Petit Arnaud , Leblanc Thierry , Bertrand Yves , Mazingue Françoise , Lutz Patrick , Vérité Cécile , Berthou Christian , Galambrun Claire , Sirvent Nicolas , Yacouben Karima , Chastagner Pascal , Gandemer Virginie , Reguerre Yves , Couillaud Gérard , Cayuela Jean-Michel
Published: October 2019

Here we report a retrospective analysis based on BCR-ABL transcript level at 3 months after the start of imatinib assessing its impact on subsequent response and outcome in children and adolescents with chronic myeloid leukemia (CML) enrolled in the French prospective trial Glivec Phase 4 (Millot F et al, JCO 2011).

Methods

44 children were enrolled in the Glivec Phase 4 Study. The median age was 11.5 years (range: 10 months-17 years). The median follow-up was 49 months (range: 16 to 83). We retrospectively analyzed the rates of complete cytogenetic responses (CCR) and major molecular response (MMR) 1 year after the start of imatinib, the progression free survival (PFS) and the overall survival (OS).

Results

At 3 months after the start of imatinib, 40/44 (91%) patients were evaluable for molecular response. BCR-ABL transcripts levels were: BCR-ABL> 10% in 15 (37%) pts and BCR-ABL<10% in 25 (63%) pts. Children and adolescents with BCR-ABL >10% at 3 months had similar Sokal score distribution but a larger spleen size and a higher leukocyte count at diagnosis compared with patients with BCR-ABL <10%. The median dose of imatinib administered within the first 3 months of treatment was similar in patients with BCR-ABL >10% and those with <10% at 3 months. Two patients progressed to blastic phase 25 and 42 months after the start of imatinib and 1 died. Both of them had a BCR-ABL transcript level of more than 10% at 3 months after the start of imatinib. A transcript level <10% correlated with a better PFS (Table 1). Patients with a BCR-ABL transcript level <10% 3 months after the start of imatinib had a higher rate of CCR and MMR 12 months after the start of imatinib compared to those with a BCR-ABL transcript level >10% (Table 1). However the difference is statistically significant only for the molecular response.

Conclusion

The children and adolescents with >10% of BCR-ABL at 3 months after the start of imatinib are characterized by a higher propensity to fail the treatment and to progress. The value of a cut-off of 10% of BCR-ABL 3 months after the start of imatinib as a reliable surrogate marker of response at 1 year and outcome remains to be determined in a larger cohort of children and adolescents.

Disclosures:

No relevant conflicts of interest to declare.
Combination Chemotherapy With Tyrosine Kinase Inhibitors Can Overcome Bcr-Abl Mutations In Acute Lymphoblastic Leukemia Or Blast Crisis CML
Authors: Benjamini Ohad , Kantarjian Hagop M , O'Brien Susan , Thomas Deborah , Ravandi Farhad , Jain Preetesh , Pierce Sherry , Borthakur Gautam , Kadia Tapan M. , Jabbour Elias , Estrov Zeev , Luthra Rajyalakshmi , Cortes Jorge E.
Published: October 2019

Background

Point mutations in the kinase domain of bcr-abl confer resistance to tyrosine kinase inhibitors (TKIs) in patients with blast phase chronic myeloid leukemia (CML-BC) and Philadelphia positive acute lymphoblastic leukemia (Ph+ ALL). In particular the presence of T315I mutation is highly resistant to most current available TKIs.
Inhibition Of Erythropoiesis By Iron Overload Is Mediated Through TGFβ Signaling
Authors: Gundabolu Krishna , Chen Huiyong , Li Huihui , Shakaladevanapura Lohith , Bhagat Tushar D. , Vallumsetla Nishant , Ginzburg Yelena , Verma Amit
Published: October 2019

Several diseases, such as β-thalassemia and myelodysplastic syndrome, are characterized by iron overload and dysfunction erythropoiesis. Multiple retrospective studies suggest that iron overload is associated with increased disease burden and shortened survival in patients. It is general consensus that iron overload has an inhibitory effect on erythroid differentiation, but direct evidence is lacking and the mechanisms underlying this phenomenon are incompletely understood. Excess iron is associated with the generation of reactive oxygen species (ROS) that result in cellular damage. We hypothesized that iron overload leads to the stimulation of myelosuppressive pathways that result in decreased erythropoiesis and resulting cytopenias. We first evaluated the effect of exogenous iron on the growth of primary human CD34+ stem cells and demonstrated that iron overload leads to decreased erythroid colony formation in vitro. Furthermore, in our in vivo experiments, we administered a total of 130 mg iron dextran IP over 20 days to C57BL/6 mice and compared these mice with PBS injected age and gender matched controls (n=5/group). We demonstrate that iron injection results in fewer circulating reticulocytes (200 vs. 310 x 109 cells/L; P=0.001) and hemoglobin (13.8 vs. 15 g/dL; P=0.0004). Using anti-CD44 and TER119 as flow cytometric markers, our data reveals a reduction in bone marrow erythroid burden (total erythroid precursors 9.6 vs. 15.3% in controls; P=0.04) with a disproportionately greater effect on orthochromatophilic erythroblasts (4.4 vs. 6.9%; P=0.02). As expected, bone marrow-derived erythroid precursors from iron loaded mice have more ROS (MFI 1497 vs. 958; P=0.05) as measured by flow cytometry, again with a disproportionately greater effect on orthochromatophilic erythroblasts (MFI 671 vs. 403; P=0.01). No effect on cell cycle was observed. To evaluate the mechanism behind iron induced suppression of erythropoiesis, we conducted a functional screen with variety of cytokine inhibitors. We observed that a specific inhibitor of the TGFβ receptor I kinase led to reversal of iron induced suppression of erythroid colonies from primary CD34+ cells in vitro. The ability of TGFβ kinase inhibitor, LY-215, in reversing iron mediated suppression was confirmed in variety of hematopoietic cell lines, resulting in the inhibition of iron induced apoptosis in these cells. Taken together, our data demonstrates for the first time that iron overload has direct suppressive effects on erythropoiesis. These effects are reversed by specific inhibitors of the TGFβ receptor I kinase and thus provide a preclinical rationale for these inhibitors in anemias associated with iron overload.

Disclosures:

No relevant conflicts of interest to declare.
Human Equilibrative Nucleoside Transporter 1 (hENT1) Expression Level Is a Potential Predictive Tool For Response To Decitabine In Patients With Myelodysplastic Syndrome
Authors: Wu Lingyun , Li Xiao , Chang Chunkang , Xu Feng , He Qi , Wu Dong , Su Jiying
Published: October 2019

Objective

The DNA methylation inhibitor decitabine is approved for the treatment of myelodysplastic syndromes (MDS). But there remains no effective factor to predict response to decitabine before therapy. Expression level of genes related to decitabine metabolism (including human equilibrative nucleoside transporter 1 (hENT1), hENT2, deoxycytidine kinase (dCK) and cytidine deaminase (CDA)) may influence the therapeutic effect of decitabine. In this study, we aimed to investigate whether the expression level of these genes could predict response to decitabine in MDS patients.

Patients and Methods

We performed Quantitative Real-Time PCR to examine expression of hENT1, hENT2, dCK and CDA prior to therapy in 60 MDS patients initially treated with decitabine. Response to decitabine and overall survival of patients treated with decitabine was analyzed grouped by the genes expression level.

Results

The patients responded to decitabine presented increased mean hENT1 expression levels compared to non-responders (0.8± 0.2 fold vs. 1.6±0.2 fold, p = 0.008). Among the 31 hENT1 high expression patients, 27(87.1%) achieved response, compared to 15/29 (51.7%) in low hENT1 patients (χ2=8.927, p=0.003). 41.9% (13/31) in hENT1 high expression patients and 10.3% (3/29) in low hENT1 patients achieved CR (χ2=7.646, p=0.006). Patients with hENT1 high expression showed longer survival time compared to low hENT1 patients (22 vs. 16 months; p = 0.027). However, the expression level of hENT2, dCK and CDA did not affect response rate and survival in MDS patients treated with decitabine.

Conclusion

hENT1 expression level is a potential predictive tool for response to decitabine in patients with MDS. High expression of hENT1 predicts prolonged survival in MDS patients treated with decitabine.

Disclosures:

No relevant conflicts of interest to declare.
The Role Of PRDM1 and Its Interacting Proteins In The Pathogenesis Of Chronic Lymphocytic Leukemia
Authors: Shukla Ashima , Chaturvedi Nagendra K , Joshi Shantaram S , Bierman Philip , Cornish Adam , Pandey Sanjit , Guda Chittibabu
Published: October 2019

Chronic Lymphocytic Leukemia (CLL) represents the most common adult leukemia in western countries, affecting approximately 10,000 individuals every year in United States of America. In order to develop effective therapeutic strategies there is a need to understand the precise molecular events associated with CLL development and progression. In an attempt to understand the process we performed transcriptome analysis of CLL cells from peripheral blood (PB) of 7 good prognosis and 8 poor prognosis patients. Additionally to validate the results further, we have utilized gene expression profiling data of CLL cells from 77 good or poor prognosis patients. Using transcriptome and gene expression profiling, we identified PR Domain Zinc Finger Protein1 (PRDM1) as one of the candidate tumor suppressor genes in CLL. PRDM1/BLIMP1 is a transcriptional repressor which is crucial for terminal differentiation of mature B cells into plasma cells. PRDM1 has been shown to promote differentiation by repressing genes essential for B cell receptor signaling and cellular proliferation. Our results demonstrated that PRDM1 expression was significantly (p < 0.05) decreased in CLL cells from poor prognosis compared to good prognosis CLL cases (Figure 1). In addition, to determine the clinical significance, the expression levels of PRDM1 were found to correlate with time to treatment in CLL patients (Figure 2). Lower expression of PRDM1 was associated (p=0.001) with shorter time to treatment in CLL (n = 40). Furthermore, using IPA analyses we identified PRDM1 interacting partners. Among those, there was increased expression of BCL2, PAX5, EHMT2, SPIB and decreased expression of BCL6, IRF4, BCR, CASP3, pFOS, TP53 and HDAC2 in CLL cells from poor prognosis patients in comparison with good prognosis. Also we have observed the differential expression of TLE1, a co-repressor molecule associated with PRDM1 and member of the Groucho family of proteins.Figure1Transcript levels of PRDM1 in Good Prognosis and Poor Prognosis.Figure1. Transcript levels of PRDM1 in Good Prognosis and Poor Prognosis.Figure2Clinical correlation of PRDM1 expression with time to treatment.Figure2. Clinical correlation of PRDM1 expression with time to treatment.

Together these results are suggestive of a role for PRDM1 as a negative regulator of CLL aggressiveness and progression. These studies warranted additional investigation to elucidate the mechanism of PRDM1 mediated CLL progression and to identify a therapeutic target.

Disclosures:

No relevant conflicts of interest to declare.
ALDH Genes and Reactive Aldehydes Play Important Roles in HSCs and Leukemia and May Be Exploited to Treat AML
Authors: Gasparetto Maura , Jordan Craig T. , Minhajuddin Mohammad , Pollyea Daniel A , Vasilou Vasilis , Reigan Philip , Humphries R. Keith , Smith Clayton A.
Published: October 2019

ALDH1A1 is expressed at high levels in normal HSCs and we previously reported that its function might involve metabolism of compounds termed reactive aldehydes. We also reported that loss of ALDH1A1 led to a compensatory increase in a related isoform, ALDH3A1, which also metabolizes reactive aldehydes. Double knockouts for both ALDH1A1/3A1 accumulate reactive aldehydes, which appear to impact a number of cellular processes including signal transduction and gene expression. As reactive aldehydes also cause DNA damage, we hypothesized that excess accumulation of reactive aldehydes may predispose to leukemic transformation of HSCs. In support of this, we found that ALDH1A1/3A1 double knockout HSCs readily form acute leukemia following transduction with a NUP98-HOXA10 fusion gene, which rarely causes leukemic transformation in wild type HSCs. Furthermore, in human AML, frequent absence of ALDH1A1 and the universal absence of ALDH3A1 was observed. A human AML cell line, Kasumi-1, was found to be ALDH1A1/3A1 deficient and to have high levels of intracellular reactive aldehydes. In addition, Kasumi-1 was highly sensitive to DNA damage and cell death following exposure to exogenous 4-HNE, a prototypic reactive aldehyde. In contrast, normal CD34+ HSCs were relatively resistant to 4-HNE. Based on these observations, we further hypothesized that treatment of ALDH1A1/3A1 deficient AMLs with clinically relevant compounds that further increase intracellular 4-HNE levels would selectively eliminate AML while sparing normal CD34+ HSCs. To test this, Kasumi-1 were exposed to a series of compounds including the pro-oxidant Arsenic tri-oxide (ATO), the sesquiterpene lactone parthenolide (PTL) and 4-HC, the active metabolite of cyclophosphamide (Cy) and a substrate of ALDH1A1. All increased intracellular 4-HNE levels and DNA damage. Exposure to combinations of 4-HC, ATO and PTL induced high levels of cell death in Kasumi-1. In contrast, Kasumi-1 cells engineered to express ALDH1A1 through lentiviral gene transfer and normal CD34+ HSCs were relatively resistant to several of these treatments. Primary ALDH1A1/3A1- AMLs were also relatively sensitive to treatment with these same compounds.

In conclusion, ALDHs and reactive aldehydes may play important roles in HSCs and leukemia and exploitation of their biology may lead to novel therapies for AML and possibly other cancers. As an initial application of this treatment strategy, we are developing a clinical trial to treat patients with relapsed/refractory ALDH1A1/3A1 deficient AML with Cy/ATO.

Disclosures:

No relevant conflicts of interest to declare.
Maintaining High Level Of Care At Outreach Sickle Cell Clinics
Authors: Hamm Jennifer , Hilliard Lee , Howard Thomas H. , Lebensburger Jeffrey D.
Published: October 2019

Introduction

Parents of children with sickle cell disease often seek care at large, university based sickle cell clinics. A major health care barrier for children in Alabama involves the cost and time of travelling to and from university based clinics. To reduce this health care barrier, the University of Alabama at Birmingham (UAB) developed The Children and Youth Sickle Cell Network(®) (CYSN(®)). This network consists of the central sickle cell clinic located at UAB and four outreach sickle cell clinics located in Montgomery (100 miles south of UAB), Opelika (110 miles southeast of UAB), Huntsville (100 miles north of UAB), and Tuscaloosa (60 miles west of UAB). The goal for these clinics is to maintain a similar level of medical care while reducing the health care barrier of transportation.

Objective

To determine if the outreach clinics provide similar care to university based clinics, we evaluated three surrogate preventive care markers to compare access to care in central vs. outreach clinics: 1) attendance rates, 2) number of patients on hydroxyurea, and 3) percent of MRIs obtained for screening of silent infarct among eligible patients.

Methods

A retrospective review of all CYSN(r) clinic visits from June 2012 to June 2013 was performed to determine clinic attendance rates. All patients on hydroxyurea were categorized by clinic location. Every patient attending CYSN(r) clinic between ages of 6 and 15 years had their medical record reviewed for completion of a screening MRI/MRA.

Results

At the central Birmingham clinic, the appointment show rate was 59.8% as compared to the Montgomery, Opelika, Huntsville, and Tuscaloosa show rates which were 57.7%, 73.1%, 59.4%, and 70% respectively. At UAB, institutional guidelines were developed for offering hydroxyurea to patients based on clinical indications and applied to all clinics. The percentage of patients on hydroxyurea therapy in Birmingham is 22.2%, while the percentages in Montgomery, Opelika, Huntsville, and Tuscaloosa are 21.5%, 32%, 21.4% and 24.4%, respectively. Finally, screening MRI/MRA to evaluate for evidence of silent cerebral infarctions is performed in Birmingham but offered to children ages 6-15 years at all sickle cell clinics. In Birmingham, 63.6% of eligible patients completed MRI/MRA screening. This percentage is similar for patients in Montgomery, Opelika, and Tuscaloosa who were screened at 66.7%, 83.3%, and 67.7% respectively.

Conclusions

Our data suggests that outreach clinics can provide similar levels of medical care for children with sickle cell disease. Sickle cell centers treating patients that must travel long distances should consider developing outreach clinics to help reduce this major health care barrier.

Disclosures:

No relevant conflicts of interest to declare.
What Is The Optimal Dose Of Methotrexate In Primary Central Nervous System Lymphoma Treatment Regimens?
Authors: Dalia Samir , Price Samantha L , Forsyth Peter , Bello Celeste M. , Shah Bijal D. , Sokol Lubomir , Sotomayor Eduardo M. , Jaglal Michael V.
Published: October 2019

Introduction

Primary central nervous system lymphoma (PCNSL) is a rare disorder with a poor prognosis. The mainstay of treatment is single agent or combination high dose (≥3.5g/m2) methotrexate (HDMTX) based regimens. There is no consensus as to which dose of HDMTX improves outcomes in patients with PCNSL but doses of MTX greater than 3 g/m2 intravenously achieve therapeutic cerebrospinal fluid (CSF) concentrations.

Purpose

To determine if there is an optimal or total dose of HDMTX in PCNSL therapies that results in improved progression free (PFS) or overall survival (OS).

Methods

Patients at Moffitt Cancer Center with PCNSL were identified using our institutional database between January 1, 2000 and September 30, 2011. Patients with complete treatment data who were treated with HDMTX were included in this study. HDMTX was defined as MTX at a dose ≥ 3.5g/m2. Patient demographics, clinical, and treatment data were collected and analyzed. Treatment information collected included the starting dose of HDMTX, IV rituximab use, MTX toxicity and clearance, cycles of MTX, and total amount of MTX administered (g/m2).

Data were analyzed using descriptive statistics and the Kaplan-Meier (KM) method was used to estimate median PFS and OS using the log rank test. P value of <0.05 was considered significant. All data was analyzed using SPSS statistical software version 21.0.

Results

A total of 51 patients were identified (Table 1).

Median PFS was 13months (0-33) and median OS 43months (29-57). The addition of IV rituximab or other chemotherapy failed to improve PFS or OS. HDMTX dose reductions or the total dose of HDMTX administered did not significantly impact PFS or OS. Similarly, when comparing dosing of HDMTX there was no significant difference in 8g/m2 versus 3.5g/m2 (PFS p=0.56, OS p=0.68), or between patients receiving 8g/m2 versus <8g/m2 (PFS p=0.77, OS p=0.6) (Figure 1). Patients receiving 8g/m2 versus those receiving <8g/m2 of HDMTX had similar baseline characteristics except for more liver function abnormalities in the 8g/m2 group.

Conclusions

Differences in initial dosing of HDMTX or total dose of HDMTX therapy did not influence outcomes in our patients with PCNSL. Dose reductions in HDMTX, addition of other chemotherapeutic agents, or rituximab were not associated with improved PFS or OS. An intriguing plateau was observed in OS in the 8gm/m2 arm despite similar PFS, suggesting that the receipt of novel therapies in the relapsed setting may contribute to OS. Multicenter collaborative clinical trials are needed to further assess the optimal initial dose of HDMTX to administer in PCNSL.

Disclosures:

No relevant conflicts of interest to declare.
Primary Mediastinal Large B-Cell Lymphoma: Investigation On The Role Of Rituximab and PET During Treatment
Authors: Zinzani Pier Luigi , Pellegrini Cinzia , Casadei Beatrice , Derenzini Enrico , Broccoli Alessandro , Gandolfi Letizia , Quirini Federica , Argnani Lisa , Stefoni Vittorio
Published: October 2019

Due to limited prospective studies, the optimal treatment of primary mediastinal large B-cell lymphoma (PMLBCL) is still a matter of debate. Third-generation MACOP-B (adriamycin, cyclophosphamide, vincristine, bleomycin, methotrexate and prednisone) regimen in combination with mediastinal radiotherapy (RT) seems to improve disease free survival of patients. In addition, the impact of additional treatment with rituximab and the role of PET are still under investigation due to controversial reported results.

As per institutional guidelines, MACOP-B plus RT was recommended in all PMLBCL patients until 2002. Aim of this report was evaluate the outcome of PMBCL patients diagnosed and treated with MACOP-B plus rituximab and consolidative mediastinal RT (30-36 Gy) after 2002. PET role was also investigated. Seventy-four patients were deemed eligible for this study (follow up of at least 2 years). Fifty patients had stage II and 24 stage IIE-IV, bulky disease was documented in 93% of patients. Median age was 34 years (range, 17-62) and 59.5% were females. All patients were evaluated by both CT and PET scan. After the final PET evaluation, PET-negative patients were observed while PET-positive patients underwent mediastinal RT.

At the end of treatment, 61 (82.4%) patients achieved a complete response (CR); 51 (68.9%) presented a positive final PET and were treated with local RT, while the other 23 (31.1%) had a negative PET. Five patients relapsed within 12 months. At 10 years, estimated overall survival was 82%, progression-free survival was 87.6% and disease-free survival (DFS) for the 61 CR patients was 90.5% (median follow-up 4 years). Regarding the DFS curve (figure 1), no statistically significant differences were observed between patients who underwent also RT (PET-positive, group 1) and patients who remained under observation (PET-negative, group 2): 90.7% (4/51 relapses) vs 90% (1/23 relapse) (p= 0.85), respectively. Comparing these results with our institutional historical series when the front-line for PMLBCL patients included only MACOP-B plus RT without any decision related to PET results (before 2002), the 10-year DFS resulted lower, i.e. 82.8%.

Although with the limitations of an observational retrospective study, the present report underlines that the additional treatment with rituximab does not change the final results in terms of CRs and DFS utilizing third-generation regimens. Moreover, the introduction of the PET-guided RT approach after MACOP-B plus rituximab allows a patient tailored strategy which reduces the use of RT and preserves clinical outcomes.

Figure 1 Figure 1.

Disclosures:

No relevant conflicts of interest to declare.
GPRC5D Is a Cell Surface Plasma Cell Marker Whose Expression Is High In Myeloma Cells and Reduced Following Coculture With Osteoclasts
Authors: venkateshaiah Sathisha Upparahalli , Bam Rakesh , Li Xin , Khan Sharmin , Ling Wen , Randal S Shelton , Yaccoby Shmuel
Published: October 2019

Identification of unique cell surface markers on myeloma cells is important for development of targeted therapies and detection of residual disease. GPRC5D is an orphan receptor reportedly expressed at high level in bone marrow (BM) aspirates or biopsies from myeloma patients. Other studies detected GPRC5D in skin and brain but not in other tissues. The aims of the study were to assess GPRC5D gene expression and cell surface localization in myeloma cells, the changes in expression of GPRC5D and other typical myeloma cell markers following coculture of primary myeloma cells with osteoclasts and the consequences of GPRC5D gene silencing on myeloma cell growth. Global gene expression profile (GEP), qRT-PCR and immunohistochemistry revealed exclusive high expression of GPRC5D in normal and myeloma plasma cells but not in B cells, MSCs, osteoclasts or BM mononucleated cells. GPRC5D expression was higher in myeloma plasma cells from newly diagnosed patients (n=698) compared to normal plasma cells (n=26, p<0.0001)). Among molecularly classified groups GPRC5D expression is higher in MS, MF and LB subgroups and lowest in CD2 and MM cell lines. Flow cytometry analysis and immunohistochemistry detected cell surface expression of GPRC5D in myeloma plasma cells while nuclear localization was also detected in certain myeloma cell lines (e.g. H929). Western blots analysis confirmed GPRC5D expression in whole cell lysate and nuclear fraction. We and other demonstrated phonotypical plasticity of myeloma plasma cells capable of altered expression of recognizable plasma cell markers (e.g. CD138, CD45) following coculture with stromal cells (Dezorella et al., 2009) or osteoclasts (Yaccoby, 2005). In coculture of primary CD138-selected myeloma cells with osteoclasts (n=8), CD138 (p<0,004), CD38 (p<0.001) and GPRC5D (p<0.007) were commonly and significantly downregulated, CD45 (p<0.02) was upregulated, and IRF4 expression was unaffected in cocultured myeloma cells compared to the control freshly obtained uncultured myeloma cells assessed by GEP and qRT-PCR. We also observed reduced expression of GPRC5D in MM cells purified from focal lesion compared to interstitial marrow in paired clinical samples (n=176, p<1.21E-09), suggesting that high activity of osteoclasts in osteolytic lesions mediates phenotypical alteration in myeloma cells. Stable knockdown of GPRC5D by 70% in CAG myeloma line using lentiviral particles containing shRNA had no effect on short-term growth of these cells assessed by MTT assay. These data indicate that GPRC5D is a novel cell surface marker for myeloma plasma cells and that its expression is reduced in dedifferentiated myeloma cells.

Disclosures:

No relevant conflicts of interest to declare.
Utility Of 9-Color, 11-Parameter Flow Cytometry For Detection Of Plasma Cell Neoplasms: A Comparison With Bone Marrow Morphologic Findings and Concurrent M-Protein Studies In Serum and Urine
Authors: Behdad Amir , Ross Charles Warren , Jacques Joshua , Kota Usha , Brown Noah A. , Keren David , Stoolman Lloyd
Published: October 2019

Background

Multiparameter flow cytometry (MFC) is a widely available laboratory platform used for primary diagnosis of mature B-cell and plasma cell (PC) neoplasms. Ongoing advances in myeloma therapeutics have drawn attention to the need for advanced laboratory methods for monitoring disease response and defining disease eradication. Recent literature indicates that achievement of remission based on sensitive MFC criteria is prognostically superior to remissions defined by older criteria based on monitoring of paraproteins in blood and urine. As such, the International Myeloma Working Group (IMWG) recommends MFC for minimal residual disease (MRD) testing during clinical trials for multiple myeloma (MM). This study validates a sensitive 9-color, 11-parameter, two-tube MFC assay suitable for both the initial diagnosis of patients with paraproteinemias and MRD monitoring in MM.

Method

We established and validated a 9-color, 11-parameter MFC assay in our laboratory. The 9-color tube contained cytoplasmic κ/cytoplasmic λ/CD45/CD38/CD56/CD138/CD19/CD117/CD20. Results of the MFC were compared to bone marrow microscopic examinations, immunohistochemical (IHC) studies and serum/urine M-protein measurements ordered on 363 samples from patients with documented or suspected PC neoplasms and IgM paraproteinemias.

Results

Total preparation time is approximately 90 minutes (“hands-on” time under 30 minutes) for up to 3 concurrent specimens. The assay is performed in two tubes (control and PC panel) with analysis of up to 1.8 x 106 total bone marrow cells/tube. The mean instrument analysis time was ∼5 minutes/tube (range 1-9 minutes). MFC detected clonal PCs in 19% and 23% of cases in which IHC or morphologic evaluation, respectively, failed to show a clonal plasma cell population. The MFC assay consistently detected clonal PC in bone marrow aspirates when serum M-protein levels were above 1g/dl. The frequency of clonal PC detection by MFC fell in concert with M-protein levels. However, in 11% of patients, MFC detected clonal PC after serum and urine immunofixation studies turned negative.

Conclusion

We conclude that the assay described herein is suitable when immunologic studies are indicated for either primary diagnosis or MRD detection in PC neoplasms. It equals or exceeds sensitivities reported in the literature and it is readily integrated into a high volume, multipurpose clinical flow cytometry laboratory.

Disclosures:

Keren: Sebia, Inc (Norcross, GA). : Consultancy.
Role Of Cytogenetic Abnormalities At Baseline and During 5-Year Follow-Up In Multiple Myeloma Patients Treated On The Total Therapy 3 Protocol
Authors: Waheed Sarah , Sawyer Jeffrey , Rosenthal Adam , van Rhee Frits , Petty Nathan M , Usmani Saad Z , Bailey Clyde , Heuck Christoph , Hoering Antje , Barlogie Bart
Published: October 2019

The presence of metaphase cytogenetic abnormalities (CA) has long been considered an adverse prognosticator in multiple myeloma (MM). An update is provided of overall survival (OS) and progression-free survival (PFS) of patients treated with Total Therapy 3 (TT3). The prognostic role of CA was assessed relative to its presence at baseline (BL) and during 2 landmarks (LM) of 2 (LM2) and 5 years of follow-up (LM5). For both LM, the following CA combinations were considered: no CA at BL and up to LM; CA at BL and till LM; CA at BL, no CA till LM; no CA at BL, CA toward LM. Patients considered for evaluation had at least one cytogenetic evaluation prior to the start of therapy and at least one cytogenetic evaluation during follow-up. In Cox modeling, only patients with at least one cytogenetic evaluation prior to baseline were considered. The time-dependent variable indicating whether patients lived to reach a certain LM, and had a CA during the time between enrollment and that LM, was considered along with several typical MM predictor variables. Results showed that, for the LM2, patients with BL-no CA/LM-no CA fared best followed by the BL-CA/LM-no CA combination, with worst outcomes seen in the remainder sharing LM-CA (Figure 1a). In case of the LM5, LM-no CA was superior regardless of BL-CA status, while LM-CA conferred equally poor OS regardless of BL-CA status (Figure 1b). On multivariate analysis, LM5-CA retained the highest HR along with GEP-70-defined high-risk MM, BL-CA and creatinine elevation. We conclude that serial CA monitoring provides a simple cost-effective tool contributing to early detection of disease relapse often without clinical progression (data not shown) that also affords the early detection of MDS-associated CA, an increasing complication of MM therapy. Data will be presented on the detection at BL of GEP features forecasting CA development during 2 and 5 years of follow up.

Disclosures:

Usmani: Celgene: Consultancy, Research Funding, Speakers Bureau; Onyx: Research Funding, Speakers Bureau.
Neutrophil Akt2 Plays a Critical Role In Heterotypic Neutrophil-Platelet Interactions During Vascular Inflammation
Authors: Li Jing , Kim Kyungho , Hahm Eunsil , Gordeuk Victor R. , Hay Nissim , Du Xiaoping , Cho Jaehyung
Published: October 2019

Platelet-leukocyte interactions on activated endothelial cells play important roles in mediating pathological thrombosis and inflammation. Heterotypic platelet-leukocyte aggregation is mediated by the interaction of two crucial receptors and counter receptors; P-selectin-P-selectin glycoprotein ligand-1 and glycoprotein Ibalpha-alphaMbeta2 integrin. In spite of extensive understanding of receptor-counter receptor interactions, it remains unclear how heterotypic cell-cell interactions are regulated under thrombo-inflammatory conditions. Using real-time fluorescence intravital microscopic analysis of Akt isoform-specific knockout (KO) mice, we have demonstrated that Akt2, but not Akt1 or Akt3, plays an important role in neutrophil adhesion to the site of TNF-alpha-induced vascular inflammation. Further, heterotypic platelet-neutrophil interactions on the activated endothelium were markedly reduced in Akt2 but not Akt1 or Akt3 KO mice. Studies with chimeric mice generated from bone marrow transplants on wild-type and Akt2 KO mice revealed that hematopoietic but not endothelial cell Akt2 regulates neutrophil recruitment and platelet-neutrophil interactions during vascular inflammation. Using in vitro reconstituted systems in which platelets and neutrophils were treated with an Akt2 specific inhibitor or cells were isolated from WT and Akt KO mice, we observed that both platelet and neutrophil Akt2 play an important role in platelet-neutrophil aggregation under shear conditions. In particular, neutrophil Akt2 was critical for membrane translocation, activation, and adhesive function of alphaMbeta2 integrin. We found that the basal phosphorylation levels of Akt isoforms are significantly increased in neutrophils and platelets of patients with sickle cell disease (SCD), which is an inherited hematological disorder with vascular inflammation and occlusion. Also, SCD patients’ neutrophils show increased alphaMbeta2 integrin activation in the absence of an agonist, in comparison with healthy donors’ cells. Inhibition of Akt2 dose-dependently reduced heterotypic aggregation of patients’ neutrophils and platelets in vitro and inhibited neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow. Our results provide important genetic and pharmacologic evidence that neutrophil Akt2 regulates alphaMbeta2 integrin function and thus plays a critical role during neutrophil recruitment and neutrophil-platelet interactions under thrombo-inflammatory conditions such as SCD.

Disclosures:

No relevant conflicts of interest to declare.
STAT3 Inhibition Promotes Potent Th1 Responses By Down Regulating Pdl-1 Expression On Tumor Cells
Authors: Vasir Baldev , Rajabi Hasan , Mills Heidi , Rosenblatt Jacalyn , Stroopinsky Dina , Luptakova Katarina , Coll Maxwell D , Kristen Palmer , Kufe Donald , Avigan David
Published: October 2019

STAT3 signaling is upregulated in cancer cells and is thought to be a critical mediator of tumor associated immune suppression. We examined the relationship of STAT3 and PDL-1, an important negative regulator of T cell immune responses via interactions with PD-1. The 5'upstream, putative promoter region of PDL-1 gene contains four potential STAT3 binding sites close to transcription start site, suggesting a possible role of STAT3 in transcriptional regulation of PDL-1. We investigated for a possible relationship between STAT3 and PDL-1 in multiple myeloma (MM-RPMI) cell line. Using chromatin immunoprecipitation assay (ChIP assay), we observed direct binding of STAT3 to the PDL-1 promoter region in MM-RPMI cells. Moreover, an enhanced occupancy of STAT3 on the PDL-1 promoter region was also observed when the cells were treated with IL-6 (20 ng/ml). Concurrent with the above observation, IL-6 treated MM-RPMI cells showed an enhanced expression of PDL-1 protein. Treatment of RPMI-MM cells with STAT3 inhibitors AG490 and CDDO-Me for 72 hrs resulted in PDL-1 expression levels of 24.5% and 15.1% respectively, compared to 97.5% for untreated control cells as determined by FACS analysis. Based on these observations, we evaluated the effect of STAT3 inhibition by exposure to small molecule STAT3 inhibitor on response to a tumor vaccine consisting of myeloma cells fused with dendritic cells (DC). DC/myeloma fusion cells were co-cultured with autologous T cells in the presence or absence of small molecule STAT3 inhibitor (CDDO-Me). Treatment of the cocultures with CDDO-Me resulted in an increased in the percentage of CD4/IFNγ (11.57±SEM 1.16; n=3) and CD8/IFNγ (23.37±SEM 2.25; n=3) expressing cells as compared to untreated cocultures (3.1± SEM 0.72; n=3) and (4.43±SEM 0.81; n=3) respectively. Moreover, we also observed a concomitant decrease in both CD4+ and CD8+ T cells expressing IL-10 or IL-4 compared to untreated cocultures. We also noted a decrease in the percentage of immunosuppressive regulatory T cells (CD4+/CD25+/FOXP3+) in treated cocultures (8.33±SEM 0.59; n=3) as compared to untreated cocultures (28.50±SEM 2.0; n=3). These findings indicate a pivotal role of inflammatory cytokine IL-6 and its downstream activator STAT3 in regulating PDL-1 expression. Thus, strategic inhibition of STAT3/IL-6 pathway and therefore down regulation of PDL-1 could enhance the potency of tumor/DC fusion cell based cancer vaccines for immunotherapeutic treatments. Furthermore, the data demonstrate that the availability of novel adjuvants offers the potential for combination therapy that may have profound effect on the potency and durability of responses to cellular immunotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Factors Influencing Peripheral Blood Progenitor Cell Yield After 2nd Mobilization With G-CSF In Allogeneic Donors
Authors: Hölig Kristina , Kramer Michael , Zimmer Kristin , Blechschmidt Matthias , Poppe-Thiede Kirsten , Bornhäuser Martin , Ehninger Gerhard
Published: October 2019

Purpose

2nd collection of Peripheral Blood Progenitor Cells (PBPC) from the same donor is increasingly required for the treatment of complications after allogeneic transplantation, but information regarding the efficacy of this procedure is still limited.

Methods

Within our program 212 allogeneic donors underwent a 2nd cycle of G-CSF-application and PBPC leukapheresis between 1996 and 2012. Demographical data and details of donation are shown in table 1. G-CSF (lenograstim) was administered at a median daily dose of 7.5µg/kg for 4.5d; leukapheresis was performed on day 5 and 6, if necessary. Peripheral CD 34+ counts and leukapheresis yields after both mobilization cycles were compared. The influence of age, sex, BMI, G-CSF schedule and the interval between the donations on the efficacy of the 2nd PBPC mobilization were analyzed, too.

Results

Median CD 34+ counts in peripheral blood were 53.2/µl at day 5 of 1st mobilization compared to 49.5/µl at day 5 of 2nd mobilization. Between 1st and 2nd donation, the median yield of CD 34+ cells/kg recipient b.w. decreased from 7.3 x 106 to 6.6 x 106 (p = 0.026, see table 2). The number of aphereses necessary in both donation episodes did not differ in the majority of donors (n=175; 82.5%). A strong correlation was observed between the amount of CD 34+ cells per recipient b.w. after the 1st and 2nd PBPC donation (r=0.89). The majority (65.4%, n=17) of donors with poor mobilization (<3 x 106 CD 34+ cells/kg recipient b.w.) after the 1st leukapheresis reached similar low CD 34+ counts after the 2nd mobilization. The interval between both mobilization cycles did not affect the results of the 2nd donation. According to an adjusted model, the other variables (age, sex, BMI and G-CSF-schedule) had no additional influence on mobilization efficacy in the 2nd cycle of G-CSF application.

Conclusions

In the largest group of healthy donors reported so far, we could demonstrate a slight reduction (about 10%) of PBPC yield after the 2nd mobilization that might not be clinically significant. Demographic variables affected the results of both mobilization episodes in the same way and the interval between them was of no significance. Our data support the assumption, that stem cell mobilization is probably determined by genetic factors and the outcome of a 2nd donation can easily be predicted by the results of the 1st one.

Disclosures:

No relevant conflicts of interest to declare.