bluuebunny/crossref_metadata_2025 huggingface dataset:
The Activity of AT9283 in Acute Myeloid Leukemia in Vitro and in the Clinic Is Karyotype Dependent.
Authors: Lyons John F , Squires Matthew S , Goodall John , Yule Murray , Ravandi Farhad , Foran James , Kantarjian Hagop M.
Published: October 2019

Abstract 4159

Aurora kinases (AK) A and B are overexpressed in a proportion of patients with acute myeloid leukemia (AML) and the level of overexpression correlates with their sensitivity to AK inhibition in vitro. We recently reported data from a dose escalation study of AT9238, a small molecule inhibitor of AKs in patients with refractory leukemias in which eight of 24 AML patients with relapsed/refractory AML achieved a 3 33% reduction in bone marrow blasts and haematological improvement. All patients had received at least one line of previous therapy. Further analysis has revealed that of the eight patients with relapsed/refractory AML that benefited from treatment with AT9283 five had a normal karyotype and the remaining three patients showed evidence of isolated abnormalities of chromosome 7, including 7q loss.

Separately, we have reported that AT9283 inhibits the proliferation and survival of AML cell lines in vitro and suggested that those cell lines with complex karyotypic abnormalities responded differently from normal diploid lines.

In these experiments AML cell lines exhibit one of two phenotypes following exposure to AT9283; rapid induction of cell death at low nM concentrations (Phenotype 1) or endo-reduplication followed by cell death at a later time point (Phenotype 2). In both scenarios treatment with AT9283 results ultimately in cell death. Cell lines with a normal karyotype tended to undergo rapid apoptosis without evidence of endoreduplication at low concentrations of AT9283. These findings provide further support for the potential importance of karyotype as a determinant of outcome in the clinical study. This is the first indication that cytogenetics might be used to predict responsiveness to Aurora kinase inhibitors in the clinic.

Disclosures:

Lyons: astex therapeutics: Employment. Squires:Astex Therapeutics, Ldt: Employment. Goodall:astex therapeutics: Employment. Yule:Astex Therapeutics Ldt: Employment. Ravandi:BMS: Consultancy, Honoraria, Research Funding.
Elderly Acute Myeloid Leukemia: A Retrospective Analysis of 93 Cases.
Authors: Du Xin , Wang Yuchun , Lu Zesheng , Weng Jianyu , Zou Xiaoli
Published: October 2019

Abstract 4163

Background

Acute myelogenous leukemia (AML) is generally looked upon as a heterogeneous group of different entities originating from the malignant transformation of a hematopoietic progenitor or stem cell. AML incidence increases with age. AML is associated with a poor prognosis, particularly in older patients. The poor prognosis in older adults is because of a combination of factors, including a larger proportion of cases with adverse-risk cytogenetics and other deleterious genetic and epigenetic changes, lower rates of achieving complete remission with intensive chemotherapy, higher risk of disease recurrence after achieving remission, greater comorbidity, inability to tolerate conventional allogeneic bone marrow transplantation regimens, and other adverse prognostic factors. The outlook for these patients remains dismal and little progress as been made in the last two decades. Current treatment of elderly AML consists of intensive chemotherapy with an anthracycline and a cytarabine. To evaluate our progress in the diagnosis, treatment and outcome of this condition, we reviewed the reports of 93 newly diagnosed acute myeloid leukaemia patients>/=55 years of age admitted to our department between 2003 and 2007.

Methods

A retrospective chart review was performed of 93 patients >/=55 years of age diagnosed with acute myeloid leukemia. The clinical efficacy was observed and the overall survival(OS) were analyzed.

Results

Median age of patients was 66 (range 55-88) years, 69 patients(74%) received a combination of an anthracycline and a cytarabine, either mitoxantrone per day on days 1, 2(MA),or 35 mg/m2 pirarubicin(TA) 7–8mg/m2 or idarubicin per day on days 1, 2 (IA). Each patient was given 100mg/m2 cytarabine intravenously per day for 7 days. And 24 patients (26%) were untreated. Total CR rate was 45% after 2 regimens. Only 3 patients happened to early death. The median period of the bone marrow depression is 19 days. The follow-up was ended at Aug 2008 year. Median survival of the treated patients was 256 days and it was significantly (P = 0.003) different from the untreated patients(66 days). Of the treated group, 7 patients were still alive, and the longest time was more than 6 years.

Conclusions

The prognosis of acute myeloid leukemia in older patients remains poor, but the anthracycline chemotherapy seems to improve the outcomes among older with AML. For this difficult patient cohort, it is a better treatment that can exert a more selective anti-leukaemic activity whilst not suppressing normal haemopoiesis or having significant systemic toxicities.

Disclosures:

No relevant conflicts of interest to declare.
PAKTM-Lx: A New Luminex*®-Based Assay to Detect and Identify Antibodies to Human Platelet Antigens (HPA) in Patient Serum.
Authors: Rathore Vipul , Meyers Matthew , Ray Bryan
Published: October 2019

Abstract 4187

Platelets express a variety of glycoproteins (GPIIb-IIIa, GPIa-IIa, GPIb/IX, GPIV, CD109 and Class I HLA). Human Platelet Antigens (HPA) represent polymorphisms of these platelet surface glycoproteins. While these polymorphisms do not alter the function of the proteins, they can become target of the immune system in mismatched individuals. Alloantibodies against HPA are involved in the pathogenesis of Neonatal Allo-Immune Thrombocytopenia (NAIT), Post-Transfusion Purpura (PTP) and refractoriness to random donor platelets. In addition to generating anti-HPA antibodies, these patients also develop antibodies to Class I Human Leukocyte Antigen (HLA). Most reference laboratories now use a combination of a sensitive screening assay such as the Platelet Immunofluorescence Test (PIFT) along with a solid phase assay for antibody identication, usually either the ELISA and Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) as well as genotyping techniques for at least HPAs 1, 2, 3, and 5. Currently, MAIPA is considered the gold standard in detection of antibodies to HPA. However, all existing methods have significant limitations in terms of sample requirements, time and labor involved. These factors are a major impediment as to how many specificities can be resolved in a rapid, efficient and reproducible manner. Development of microbead-based assays like the xMAP (Luminex®) has provided a technology that can resolve these questions. GTI Diagnostics® and Gen-Probe have developed PAKTM -Lx, a bead-based assay for the Luminex® platform to detect and determine the specificity of anti-HPA antibodies. PAKTM -Lx is capable of resolving the specificities of antibodies to HPA 1, 2, 3, 4 and 5 in addition to detecting antibodies against GPIV and HLA Class-I antigens. Furthermore, it is capable of detecting antibodies to multiple HPA and HLA with only 10 μl of sample. With a total time of less than two hours from addition of samples to end results, PAKTM -Lx is sensitive, rapid and simple to perform. Focused, flexible multiplexing in the range of 1 to 100 analytes provided by Luminex® technology meets the needs to expand the panel to include more HPA combinations, thereby reducing sample requirements, labor and costs.

Disclosures:

No relevant conflicts of interest to declare.
Comparing Efficacy of Apheresis and Whole-Blood Derived Platelets Transfusions in Acute Leukemia Patients Who Undergo Chemotherapy without Remission.
Authors: Chen Qin-fen , Li Pei , Xie Yi
Published: October 2019

Abstract 4189

Objective

To determine the efficacy between apheresis platelet concentrates(APCs) and platelets (PLTs) derived from whole blood(WBD) transfusion in acute leukemia patients receiving chemotherapy without remission.

Methods

All patients received same ABO group and leukocyte depletion PLTs. According to the platelet count before and after transfusion, we used percent platelet recovery(PPR) and corrected count increment(CCI) at 24 hours post-transfusion to analysis the outcomes.

Results

A total of 130 patients between the ages of 14 and 80 received 771 times PLTs transfusion. 85 patients were males, and 45 were females. There were 596 times APCs and 175 times WBD. In APCs, the platelet counts were 11.97±10.43×109/L and 24.68±18.63×109/L pre- and post-transfusion(P<0.001). In WBD, the platelet counts were 11.06±7.58×109/L and 22.88±18.00×109/L pre- and post-transfusion(P<0.001). But there was no significant difference in the platelet count between the two types of platelet produces transfusion. According to PPR, if PPR<20% is considered platelet transfusion refractoriness(PTR), the incidence rates of PTR in APCs and WBD were 46.98% and 49.71%, respectively. According to CCI, if CCI<4.5×109/L is considered PTR, the incidence rates of PTR were 42.95% and 46.86%, respectively. The incidence rate of PTR was no significant difference in PPR and CCI between the two PLTs products.

Conclusion

For the patients of acute leukemia without remission, the incidence rate of PTR was high, even if they received same ABO group and leukocyte depletion products. The efficacy was not significant difference between APCs and WBD transfusions.

Disclosures:

No relevant conflicts of interest to declare.
Poor Hematopoietic Stem Cell Mobilizers in Multiple Myeloma:A Single Institution Experience.
Authors: Ruiz-Delgado Guillermo J. , Lopez-Otero Avril , Hernandez-Arizpe Ana , Ramirez-Medina Aura , Ruiz-Arguelles Guillermo J.
Published: October 2019

Abstract 4237

High-dose chemotherapy rescued by autologous peripheral blood stem cell transplantation is considered standard of care for patients with multiple myeloma. Hematopoietic stem cell mobilization is accomplished by administration of hematopoietic growth factors combined or not with myelosuppressive chemotherapy. In a single institution, in a group of twenty eight myeloma patients deemed eligible for autologous transplant, stem cell mobilization was attempted using only filgrastim: Twenty six individuals were given 31 autografts employing one to four (median three) apheresis sessions, to obtain a target stem cell dose of 1 × 106 CD34 viable cells / Kg of the recipient. The median number of grafted cells was 7.56 × 106 CD34 viable cells / Kg of the recipient; the range being 0.92 to 14.8. By defining as poor mobilizers individuals in which a cell collection of less than 1 × 106 CD34 viable cells / Kg was obtained, a subset of eight poor mobilizers was identified; in two patients the autograft was aborted because of an extremely poor CD34 cell yield (less than 0.2 × 106 CD34 viable cells / Kg of the recipient) after four apheresis sessions. The long-term overall survival of the patients grafted with more than 1 × 106 CD34 viable cells / Kg was better (80% at 80 months) than those grafted with less than 1 × 106 CD34 viable cells / Kg (67% at 76 months). Methods to improve stem cell mobilization are needed and may result in obtaining better results when autografting multiple myeloma patients.

Disclosures:

No relevant conflicts of interest to declare.
Sezary Syndrome Aberrant Expression of CD26 On CD4+, CD25+, FOXP3+ T Lymphocytes and Prognostic Significance.
Authors: Corinaldesi Giorgio , Corinaldesi Christian
Published: October 2019

Abstract 4244

The Sezary syndrome (SS) is a variant of cutaneous lymphoma (CTLC) the cell is a malignant pleomorphic CD4+/CD25+/FOXP3+ T cell with a convolute-cerebriform nucleus, a cytogenetic evidence of an abnormal clone were observed in all patients MTAP (17p13.1-TP53), SMARCE (17q21, 1q21.3- STAT5A/STATB), DULLARD (9p21.3-CDKN2A/CDKN2B), M13930 (8q24.21-C-MYC/PLEC1), expression profiles showed upregulation of genes involved in TNF-signaling pathaway, and decreased expression of tumor suppressor genes such as TGF-beta receptor II, while TWIST and EPHA4; BCL-2, and TEX10 were overexpressed. Aberrant expression of GATA -3, TRAIL, T-plastin, CD1D are present in more advanced stages and correlate with disease progression. The most characteristic symptons include a persistent xerosis, erythematous patches, pruritus, ectropion, alopecia, dystrophic nails, palmoplantar keratoderma lymphadenopathy, hepatosplenomegaly, and sometime fungal infection or colonization with staphylococcus aureus. The loss of CD26,and a decreasing of CD4+CD7- expression,and an increasing of CD25+(alfa-chain of IL-2 receptor) FOXP3+ Tregs is a costant feature; the association with the circulating aneuploid cells showed high expression of skin manifestation, disease progression and poorest prognosis.

Disclosures:

No relevant conflicts of interest to declare.
Imatinib Increases Cytotoxicity of Melphalan and Their Combination Allows An Efficient Killing of CML Cells.
Authors: Giallongo Cesarina , Cava Piera La , Tibullo Daniele , Parrinello Nunziatina , Guagliardo Provvidenza , Fabro Vittorio Del , Stagno Fabio , Chiarenza Annalisa , Consoli Carla , Spina Paolo , Vigneri Paolo , Palumbo Giuseppe A , Di Raimondo Francesco
Published: October 2019

Abstract 4254

BCR/ABL-positive cells are relatively resistant to chemotherapy and, in order to evaluate the effect of Imatinib (IM) in reverting drug-resistance, we evaluated on K562 the toxicity of 1 h exposure to cytosine arabinoside (ARA-C) 20 μM, hydroxyurea (HU) 100 μM, and melphalan (MEL) 20 μM, after a pre-treatment of 24 h with 1 μM IM. The doses of the drugs were similar to that achieved in the plasma after standard chemoterapeutic treatment. Cell viability was evaluated by ATP-lite at 24, 48 and 72 hs from beginning of drug-free condition. The combinations of IM plus MEL induced the highest cytotoxicity (P<0,001 at 24, 48 and 72 hs vs MEL alone) indicating that pre-treatment with IM increased K562 exposition to the genotoxic damage of MEL. We next analyzed effects on cell cycle and DNA damage by alkaline comet assay induced by this drug combination and we observed that DNA damage peaked at 48 h with IM/MEL combination. In addition, flow cytometry analysis showed that IM/MEL combination reduced the cell accumulation in G2/M phase induced by MEL (P<0.001 vs MEL), thus reducing the ability to DNA repair and recovery.

These data indicate that inhibition of BCR/ABL activity by IM increased cell cytotoxicity of MEL by reducing the effectiveness of the DNA-repair pathways and decreasing the time for DNA repair at the G2/M checkpoint..

To ascertain that these results were linked to BCR/ABL inhibition, TonB.210, a cell line where the BCR-ABL expression is inducible by doxycycline (DOX), were treated in the same conditions. Only TonB.210 cultured with DOX were insensitive to MEL while IM/MEL combination reverted these drug resistance (P<0,001).

In the final step we studied the sequential association IM/MEL on the proliferative potential of myeloid progenitors of 6 CML patients at diagnosis. IM/MEL combination increased the reduction of the overall number of colonies in comparison to IM alone (P<0.05 vs IM). In addition, the analysis on CFU-GM and BFU-E colonies by qRT-PCR demonstrated that the IM/MEL combination led to the highest reduction in the number of BCR/ABL positive colonies (P<0.01 vs IM).

Therefore, our data indicate that the pre-inhibition of BCR/ABL activity by IM increases the toxicity of MEL and allows an efficient killing of human leukemic cells, thus suggesting new therapeutic combinations for CML patients

Disclosures:

No relevant conflicts of interest to declare.
High Incidence of Radiation Induced Cavernous Hemangioma (RICH) in Long Term Survivors Who Underwent BMT with Radiation Therapy During Childhood or Adolescence.
Authors: Koike Takashi , Yanagimachi Noriharu , Yabe Hiromasa , Yabe Miharu , Morimoto Tsuyoshi , Shimizu Takashi , Ishiguro Hiroyuki , Takakura Hiromitsu , Ando Kiyoshi , Kato Shunichi
Published: October 2019

Abstract 4292

INTRODUCTION

Radiation induced cavernous hemangioma (RICH) is a late complication of cerebral radiation therapy. An increased number of long term surviving blood and marrow transplantation (BMT) recipients have recovered from their primary disease but are at risk of RICH.

METHODS

We investigated 66 patients who underwent BMT during childhood or adolescence. We evaluated RICH numbers, size, location and their annual changes. Furthermore we developed scoring system of RICH in order to classify severity. MRI of the brain was performed annually for 5 to 27 years after BMT, including gradient-echo sequence (T2* weighted image). RICH SCORE 1-4 is designated as mild, 5-9 as moderate and 10 or more as severe.

RESULTS

Twenty-five patients (37.9%) was diagnosed RICH. The age at the time of the diagnosis was 11-40 years old (median 27 years old). The age at the time of BMT was 1-22 years old (median 9 years old). The period from BMT to diagnosis was 10-24 years (median 16 years). All cases received TBI as conditioning of BMT and/or cranial radiation (CR) prior to BMT as treatment of primary disease. RICH was found in 25/48 (52%) who received TBI and/or CR, and was not found in any of 18 patients without radiation therapy to the brain. Total dose to the brain was 10-36 Gy. Clinical manifestations were present only in four cases. RICH SCORE ranged 1-18 points (median 4 points). Small RICHs tended to be recognized only by T2* weighted image, but not by routine imaging methods. Classification of the severity was mild in 13 patients, moderate in 8 patients and severe in 4 patients. Severity was correlated with higher radiation dose and/or with younger ages at transplantation. RICH SCORE increased yearly in 7 of 25 patients. One case developed giant RICH more than 40mm as shown in the attached image.

CONCLUSION

High incidence of RICH was found in long term survivors who underwent BMT with radiation therapy. Since all of those patients did not show RICH before BMT and all positive patients had a history of radiation therapy to the brain, the cause of RICH in those patients was considered to be radiation. Careful and long term evaluation with MRI including T2* weighted image is necessary in BMT recipients who received radiation therapy prior and/or during BMT.

Disclosures:

Ando: Alexion: Research Funding.
G-CSF Mobilized Versus Conventional Donor Lymphocytes in the Treatment of Hematological Malignancies.
Authors: Abbi Kamal K.S. , Ehmann W. Chritopher , Talamo Giampaolo , Lucas Kenneth , Carraher Michelle , Rybka Witold , Claxton David F.
Published: October 2019

Abstract 4301

Introduction

Donor lymphocyte infusions (DLI) can reverse relapse of hematologic malignancy following allogeneic hematopoietic stem cell transplant (HSCT) in some cases. G-CSF is used to mobilize progenitor cells for transplantation and at our center excess cells have been cryopreserved and subsequently used as DLIs. Little is known regarding the effectiveness of G-CSF mobilized cells as DLIs in the treatment of hematological malignancies. We conducted a retrospective study comparing the overall survival in patients treated with G-CSF mobilized vs. conventional DLI for hematological malignancies.

Patients and Methods

After IRB approval, we conducted a retrospective chart review of 67 patients who received DLI treatment of management of hematological malignancies.

Results

Mean age was 48.5 (range 2-78). 41 (62%) patients had AML and 26 (38%) had other hematological malignancies. Patients received 1-4 DLIs each; median was 1 DLI per patient. Median no. of CD3+ cells infused was 107/kg body weight (range 1- 265). 17 (25%) patients received a total of 32 G-CSF mobilized DLIs. 50 (75%) patients received 77 conventional DLI. 20 (29%) patients were treated with immunosuppressive agents at the time of DLI and 49 (73%) received chemotherapy in the 90 days prior to DLI. DLIs were administered for relapse or residual disease post-transplant in 62 patients. 5 patients received 7 DLIs because of poor engraftment. In total 38(56%) patients have died. Death was malignant disease related in 31/38(82%) patients, and caused by graft versus host disease (GVHD) in 5/38 (13%). GVHD deaths were 2/17 in the G-CSF and 3/50 in the conventional group (NS). Median survival after DLI was 213 days for the 67 patients and was similar for GCSF and conventional groups. 19/67 patients survived more than 1 year from the initial DLI. Death and GVHD were not predicted by diagnosis, donor type (related of unrelated), use of immunosuppressives at the time and DLI or number of infused donor CD3 cells. Time to first evidence of GVHD was similar between the two groups (p=0.35). Kaplan-Meier estimates of survival suggested that G-CSF mobilized DLI, while not statistically different from conventional DLI, showed a trend towards a survival advantage (p=0.08). This analysis is shown in the figure below.

Conclusion

In this series of poor prognosis hematological malignancies, DLI was clearly lifesaving in a number of patients. G-CSF mobilized DLI appears to be at least as effective as conventional DLI for the treatment of patients relapsing after allogeneic blood stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Alternative Donor Hematopoietic Stem Cell Transplantation for Children with Severe Aplastic Anemia.
Authors: Park Meerim , Koh Kyung Nam , Bae Keun Wook , Lee Mee Jeong , Im Ho Joon , Seo Jong Jin , Moon Hyung Nam
Published: October 2019

Abstract 4348

Background

Hematopoietic stem cell transplantation (HSCT) from matched sibling donor is the standard first-line treatment for children with severe aplastic anemia (SAA). However, the management of SAA lacking a suitable donor remains a great challenge. For those children, HSCT using unrelated donor or mismatched related donor could be a therapeutic alternative. The purpose of this study is to evaluate the outcome in children with SAA who received HSCT from donors other than matched sibling.

Patients and Method

Between March 2003 and July 2009, 17 patients received HSCT from alternative donors (AD) at Asan Medical Center. We reviewed their medical records and analyzed their transplant-related parameters and outcome.

Results

Of a total of 17 patients, 11 were male and the median age at HSCT was 9.0 years, ranging from 3.0 to 16.7 years. Four patients had Fanconi anemia and 13 had acquired SAA including 2 who developed SAA after liver transplantation. Donors included unrelated bone marrow (U-BM) in 5, unrelated peripheral blood (U-PB) in 6, unrelated cord blood (U-CB) in 2 and related haploidentical peripheral blood (H-PB) in 4. Of 17 patients, 15 (88%) achieved sustained engraftment. Of 15 with engraftment, only 1 patient who received HSCT from U-CB died of severe GI GVHD and the other 14 patients remain on stable normal counts without transfusion support. All 2 patients (1 U-BM, 1 H-PB) who failed to engraft were dead despite DLI or 2nd HSCT. With a median follow-up of 31.9 months, the Kaplan-Meier estimated overall survival at 2 years was 76.6%.

Conclusion

In children with SAA, HSCT from AD including haploidentical family donor could be considered as a treatment option if the patients have no matched sibling donor. Given the limitation of this study such as small number of patients and short follow-up period, further trial will be necessary.

Disclosures:

No relevant conflicts of interest to declare.
Conditional Expression of Heterozygous or Homozygous Jak2V617F From Its Endogenous Promoter Induces a Polycythemia Vera-Like Disease.
Authors: Akada Hajime , Yan Dongqing , Zou Haiying , Hutchison Robert E. , Mohi M. Golam
Published: October 2019

Abstract 437

A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in a majority of patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). However, the contribution of JAK2V617F in these three clinically distinct myeloproliferative disorders (MPDs) remained unclear. To investigate the actual role of JAK2V617F in the pathogenesis of MPDs, we generated an inducible Jak2V617F knock-in mouse, in which the expression of Jak2V617F is under control of the endogenous Jak2 promoter. Expression of heterozygous Jak2V617F evoked all major features of human PV, which included marked increase in hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, splenomegaly, reduced serum levels of erythropoietin (Epo) and Epo-independent erythroid colonies. Homozygous Jak2V617F expression resulted in a more severe form of PV associated with markedly elevated leukocytosis, neutrophilia and thrombocytosis, and a majority of these mice succumbed due to massive cardiac thrombosis. Activation of Stat5, Akt and Erk was significantly enhanced in erythroblast cells expressing homozygous Jak2V617F compared to heterozygous Jak2V617F, suggesting that the degree of activation of downstream signaling pathways would be affected by the Jak2V617F gene dosage. This may also partly explain the severe phenotype in mice expressing homozygous Jak2V617F. We conclude that heterozygous Jak2V617F is sufficient to cause PV, whereas homozygous Jak2V617F increases the severity of PV disease and the risk of thrombosis. Our results also provide strong evidence that Jak2V617F gene dosage does not play a defining role in determining PV versus ET phenotype in this mouse model of MPD.

Disclosures:

No relevant conflicts of interest to declare.
Relationship Between Expression of TLRs and Disease Activity in CLL.
Authors: Barcellini Wilma , Zaninoni Anna , Imperiali Francesca Guia , Reda Gianluigi , Orofino Nicola , Cortelezzi Agostino , Zanella Alberto
Published: October 2019

Abstract 4379

Background

Toll-like receptors (TLRs) are major agents of innate immunity and initiators of adaptive immune response by acting as costimulatory signals for B cells and inducing maturation, proliferation and antibody production after pathogen recognition. They are also involved in the self-antigen recognition and could play a role in autoimmune phenomena. It is well known that chronic lymphocytic leukemia (CLL) is characterized by an increased incidence of autoimmune phenomena and immunodeficiency, which can greatly influence the disease outcome leading to a variable clinical course.

Aims

To evaluate the correlation between the gene expression of TLRs in 41 patients with CLL (mean age±SD 66±18 years, range 42-90, 15 female and 26 male), the clinical course of the disease and the expression of prognostic factors (mutational status of IgVH region, CD38 and ZAP70 expression, and cytogenetic alterations).

Methods

The gene expression of TLR4, TLR9, and TLR10 was studied. Total RNA was extracted from B cells of patients and controls (pool of 10 healthy donors), cDNA was synthesized, and real-time PCR was performed. For each reaction 50 ng of cDNA were mixed with 1.25μl of TaqMan primer/probe set and 10.25μl of TaqMan Universal Master Mix. The TLR4, TLR9, and TLR10 expression was normalized according to GAPDH as an internal control gene and it was expressed as percentage of control.

Results

TLR4 gene expression was significantly lower in CLL patients compared to controls (18±3%, mean±SE) while TLR9, and TLR10 gene expression was higher (3457±500% and 2897±440%, respectively). Moreover, considering “progressive” CLL patients (n=20), TLR4 gene expression was significantly lower compared with “indolent” ones (n=21) (10.2±2.2% vs 24.9±4.9%, p=0.01). Patients with unmutated IgVH expression and unfavorable cytogenetic alterations (mostly 11q-) showed even more reduced TLR4 gene expression, but the small numbers did not allow statistical evaluation. TLR4 gene expression was lower in patients with infective diathesis, although not significantly (11.5±2.9% vs 20.0±3.9).The 6 “progressive” CLL patients with autoimmune diseases (hemolytic anemia, Evan's syndrome and phemphigus) showed significantly higher TLR4 gene expression compared with “progressive” patients without autoimmune phenomena (17.7±4.4% vs 6.9±2.1%, p=0.02), possible expression of activated immune system.

Conclusion

The reduced TLR4 gene expression in CLL patients with “progressive” disease, unmutated IgVH status, and unfavourable cytogenetic alterations is consistent with a reduced ability to a proper immune response to Gram-negative bacterial lipopolysaccarides and consequent increased susceptibility to infections.

Disclosures:

No relevant conflicts of interest to declare.
Applicability of a New Prognostic Scoring System in Myelodysplastic Syndromes. MDS Study Group of Hematology.
Authors: Flores Gabriela , Santos Maria Isabel , Alfonso Graciela , Cabrejo Maria , Prates Maria Virginia , Iastrebner Marcelo , Fantl Dorotea Beatriz , Arbelbide Jorge , Campestri Reinaldo , Penchasky Diana , Cardenas Paula , Sackmann Federico , Garcia Juan , Nucifora Elsa , Basquiera Ana , Pavlovsky Astrid , Pavlovsky Miguel Arturo , Fernandez Isolda , Viñuales Susana
Published: October 2019

Abstract 4838

Background

Myelodysplastic Syndromes (MDS) are so heterogeneous, that new prognostic scoring systems are being continuously developed to help choose the best treatment strategy. The International Prognostic Scoring System (IPSS) excludes secondary MDS, prior therapy and Chronic Myelomonocytic Leukemia (CMML) with leucocytosis. Recently, a new risk model has been published by Kantarjian et al. (Cancer 2008; 113; 6; 1351) that is applicable to all MDS patients.

Aims

To validate this new risk model in our MDS population.

Methods

We analyzed 253 patients reported from 15 centers in our country from Jan 2007 through Jun 2009. We took into account age, performance status (PS), hemoglobin, platelets, leucocytes, bone marrow blasts, karyotype, and transfusion requirements. The new model divided patients into 4 prognostic groups: Low Risk (LR): 0-4; Intermediate-1 (I-1): 5-6; Intermediate-2 (I-2): 7-8; and High Risk (HR): ≥9. We assessed the prognostic impact of this New Risk Model and compared results to IPSS. Mortality Rate and AML progression risk were analyzed.

Results

164 patients were evaluable, mean age 69 (R 21-92), primary MDS: 144, and secondary MDS: 20. Risk Group assessment for the new score: LR (34%), I-1 (31%), I-2 (18%), and HR (17%). The mean Follow-up was 22 months;

Results

are shown in table and graphic.

Conclusion

Even though our sample is not considerably big, and follow-up is short, we confirmed 4 categories with different prognosis and found a close correlation with Mortality Rate. This New Scoring System allowed us to identify Low Risk IPSS patients with different outcomes, highlighted the importance of adding new prognostic factors like age and performance status, refined the cut-offs of thrombocytopenia and anemia, and recognized the adverse impact of prior transfusions needs.

Graphic

Disclosures

No relevant conflicts of interest to declare.
Removal of Autologous Activated CD4 Positive T Lymphocytes Also Result in Increased Colony-Forming Units (CFUs) in Patients with Myelodysplastic Syndromes.
Authors: Zhang Zheng , Li Xiao , Zhang Qingqiao , He Qi , Xu Feng , Wu Lingyun
Published: October 2019

Abstract 4862

Autologous activated CD4+ T lymphocytes (CD4+/CCR5+ double positive cells) in BMNC of 29 MDS patients were depleted or plused in in vitro cultures. That cultures with removed CD4+/CCR5+ cells exhibited significantly increased CFU generation. Conversely, the 4-fold CD4+/CCR5+ T lymphocyte-plused BMNC exhibited no colonies at all in in vitro cultures. After CD4+/CCR5+ depleted in vitro cultures, the clonal cells increased in patients with chromosome 5q– or 20q–, kept almost unchanged in patients with trisomy 8, but dramatically decreased in patients with monosomy 7. In addition, after the removal of CD4+/CCR5+ T cells, better CFUs generation were found in those patients who were with higher BM Th1 (CD4+/IFNγ+) cells or hypocellularity, or bearing DR15 allele. We concluded that selective removal of autologous activated CD4+ T-cells can increase the generation of CFUs, while the increased CFU consisting of residual normal hemopoiesis or clonal hemopoiesis kept unconcluded.

Disclosures

No relevant conflicts of interest to declare.
Stable MAGEC1/CT7 Inhibition by Short Hairpin RNA (shRNA) Does Not Affect Tumor Cell Proliferation and Invasive Potential in Sko-007 Multiple Myeloma Cell Line.
Authors: de Carvalho Fabricio , Camargo Anamaria A. , Costa Érico T. , Masotti Cibele , Andrade Valéria C.C. , Strauss Bryan E. , Caballero Otávia L. , Colleoni Gisele W.B.
Published: October 2019

Abstract 4888

Introduction

MAGEC1/CT7 gene encodes for a cancer testis (CT) antigen frequently expressed in multiple myeloma (MM) and may be a potential target for immunotherapy in this still incurable disease. This CT gene expression is restricted to malignant plasma cells and may be related with tumor progression, since it seems to play a role as a “gatekeeper” gene for other CT antigens and can be associated with a more aggressive phenotype. However, the exact function of this protein for tumor biology is not yet understood.

Objective

The aim of this study is to elucidate the role of MAGEC1/CT7 in the control of cellular proliferation, DNA synthesis and invasive potential of Sko-007 myeloma cell line.

Material and Methods

We used a short hairpin RNA (shRNA) specific for MAGEC1/CT7 gene that was previously inserted in the pRS (pRETROSUPER) retroviral vector. The pRS-shRNA-MAGEC1/CT7 construct was co-transfected with pCL-amphotropic packing vector in 293T cells to produce virus particles. Sko-007 myeloma cell line was transduced and selected for stable expression of shRNA-MAGEC1/CT7. Expression of MAGEC1/CT7 was analyzed by Western Blot and Real Time PCR (RQ-PCR). Functional studies included cell proliferation, 3H thymidine incorporation and invasion potential, using matrigel-coated transwell filters.

Results

Sko-007 was chosen to perform the initial functional studies since the basal MAGEC1/CT7 gene expression level was higher in this cell line when compared with the other myeloma cell lines (U266 and SK-MM-2). Sko-007wt (wild type) cell was divided into three derivatives, Sko-007ev (empty vector), Sko-007shMAGEC1/CT7Δ (antisense strand deleted – GC bases) and Sko-007shMAGEC1/CT7. MAGEC1/CT7 expression was approximately five times down regulated in Sko-007shMAGEC1/CT7 when compared with Sko-007wt, Sko-007ev, and Sko-007shMAGEC1/CT7Δ cells by RQ-PCR. Sko-007shMAGE1/CT7 had an 80% decrease in MAGEC1/CT7 protein expression when compared with Sko-007wt, Sko-007ev and Sko-007shMAGEC1/CT7Δ by Western Blot. Our preliminary functional assays did not show any change in cell proliferation and DNA synthesis when Sko-007shMAGEC1/CT7 was compared with Sko-007wt, Sko-007ev and Sko-007shMAGEC1/CT7Δ. Indeed, Sko-007 cell line has invasive potential, but no change was observed in Sko-007shMAGEC1/CT7 or other cells (Sko-007wt, Sko-007ev and Sko-007shMAGEC1/CT7Δ).

Conclusions

Expression of MAGEC1/CT7 mRNA could be inhibited by the shRNA strategy, but the concomitant reduction of its protein level did not seem to have impact in proliferation, DNA synthesis or in invasive potential of Sko-007 MM cell line (Supported CNPq and LICR São Paulo Branch).

Disclosures

No relevant conflicts of interest to declare.
Antitumor Effects and Mechanisms of Curcumin On Human Multiple Myeloma Cell Lines.
Authors: Bai Qingxian , Liu Qifa
Published: October 2019

Abstract 4896

BackgroundF

Multiple myeloma(MM) is a malignant plasma disease, which is characterized as high relapse rate and high resistance to chemotherapy. Curcumin is a polyphenol derived from the rhizome of Curcuma spp. It possesses diverse pharmacologic actions, such as antitumor, anti-inflammatory,anti- oxidation properties .Curcumin has the property of inhibit multiple tumor cell lines, in which included multiple myeloma cell. The real mechanism is not completely clear yet. We explored the mechanisms of curcumin on human multiple myeloma cell lines (RPMI8226 and H929), and investigated whether the combination of curcumin and adriamycin(Adr) has a synergistic effect.

MethodsF

The effect of curcumin on proliferation of RPMI8226 and H929 was observed with MTT assay. The synergetic effect of curcumin and Adr was analyzed by median-effect principle. Cell cycle distribution and apoptosis were studied with flow cytometry. Expression of surviving, bcl-2, bax mRNA was detected by RT-PCR.

ResultsF

Curcumin could inhibit the proliferation of RPMI8226 and H929 cells in a time- and dose-dependent manner. The IC50 values for RPMI8226 and H929 cell line were 12.15 μmol/L,17.24μmol/L respectively. The combination of curcumin and Adr showed synergistic effect even at low concentration of Adr. Apoptotic ratio of treated cells was significantly higher than untreated controls (36.9% vs 10.6%, p<0.05). Cells treated with curcumin showed cell cycle arrest at G2/M phase. Curcumin upregulated expression of survivin, bcl-2, while bax mRNA was significantly downregulated.

ConclusionF

Curcumin could suppress the proliferation of multiple myeloma cells and induce apoptosis. Adr combining with curcumin can show synergistic effect at low concentration of Adr. The mechanism of curcumin's antitumous effect might be related to down-regulation of surviving, bcl-2 mRNA and up-regulation of bax mRNA.

Disclosures

No relevant conflicts of interest to declare.
Localized AL Amyloidosis of the Breast: A Case Series.
Authors: Charlot Marjory , Seldin David C. , O'Hara Carl , Skinner Martha , Sanchorawala Vaishali
Published: October 2019

Abstract 4906

AL amyloidosis is characterized by widespread, progressive deposition of fibrillar amyloid protein derived from monoclonal immunoglobulin light chains, leading to organ failure and death. This disease is typically systemic, however, it can occur as a localized form. In localized amyloidosis, the deposits occur near the site of synthesis of the precursor protein and in some cases, plasma cells have been demonstrated histologically adjacent to the deposits. For unknown reasons, the tracheobronchial tree is the most common site for localized AL amyloidosis. Localized AL amyloidosis of the breast is a rare entity that has been described in the literature in isolated case reports. It can present as a palpable mass or as calcifications on routine screening mammography. We report here a case series of seven women (median age 63 years, range 46 to75) seen and evaluated at Boston University Medical Center from 1990-2008. We evaluated 1502 new patients with AL amyloidosis in this time period, making the incidence of localized AL amyloidosis of the breast to be 0.5% at a single referral center. All seven patients had abnormal screening mammography with calcifications, and biopsies that revealed Congo red positive amyloid deposits. Histologically, the amyloid deposits appeared as amorphous material in the stroma around the ducts and lobules in most patients; one patient had amyloid deposits in the ducts only, but not in the stroma. None of the patients had clinical or laboratory evidence of other organ involvement, all had negative Congo red staining of an abdominal fat pad aspirate, and all had a negative work up for a plasma cell dyscrasia or circulating paraprotein. The patients were treated with local excision of the regions of calcification or lumpectomy. Three out of seven patients underwent routine follow up within 6-12 months from the time of diagnosis with no evidence of disease recurrence or progression to systemic AL amyloidosis. One out of seven patients had bilateral and recurrent amyloidosis of the breasts and was found to have an associated stage I invasive ductal adenocarcinoma that was treated with lumpectomy and radiation. In summary, breast amyloidosis is rare, is not associated with a systemic plasma cell dyscrasia or amyloidosis in other organs, and can be treated surgically.

Disclosures

No relevant conflicts of interest to declare.
Carfilzomib Exerts Anti-Neoplastic Activity in Waldenstrom Macroglobulinemia.
Authors: Sacco Antonio , Roccaro Aldo M. , Aujay Monette , Ngo Hai , Azab Feda , Azab Abdel Kareem , Melhem Molly R , Runnels Judith M. , Quang Phong , Demo Susan , Ghobrial Irene
Published: October 2019

Abstract 4916

Introduction

Proteasome inhibition represents a valid therapeutical approach in several tumors and its use has been validated in Waldenstrom's macroglobulinemia (WM), where single-agent Bortezomib has been successfully tested in phase 2 clinical trials. Nevertheless, a significant fraction of patients relapse, or develop significant toxicity due to high toxicity in non-transformed cells. Therefore preclinical evaluation of new proteasome inhibitor with a more selective inhibition of neoplastic cells is needed in order to increase efficacy and improve patient outcome. We tested Carfilzomib, a tetrapeptide epoxyketone selective inhibitor of the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in WM.

Methods

WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL) were used. Expression of imunoproteasome and constitutive proteasome subunits (beta1, beta2, beta5; LMP2, MECL1, LMP7) were detected primary WM cells and cell lines by an ELISA-based assay. Cytotoxicity and DNA synthesis were measured by thymidine uptake and MTT, respectively. Cell signaling and apoptotic pathways were determined by Western Blot. Determination of the additive or synergistic effect of drugs combination was calculated using the CalcuSyn software based on the Chou-Talalay method.

Results

Primary CD19 bone-marrow derived WM cells express higher level of the immunopreoteasome as compared to the constitutive proteasome. Carfilzomib inhibited the chymotrypsin-like activity of both the immunoproteasome (LMP7) and the constitutive proteasome (beta5) and in WM cells, in a dose-dependent manner; leading to inhibition of proliferation (IC50: 5nM; 48h) and induction of cytotoxicity (IC50: 7.5nM; 48h) in WM cells. Carfilzomib mediated apoptosis in WM by increasing PARP-, caspase-9- and -3-cleavage; as well as by inducing activation of c-jun-N-terminal kinase and ER-stress in a dose-dependent manner. Moreover, combination of Carfilzomib and bortezomib induced synergistic cytotoxicity in WM cells, as shown by enhanced PARP-, caspase-9- and -3-cleavage; and synergy in inhibiting the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome.

Conclusion

Taken together, these findings provide the pre-clinical rational for testing Carfilzomib in Waldenstrom Macroglobulinemia.

Disclosures

Aujay: Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.
Continuous Infusion Cyclophosphamide as Salvage Therapy for Relapsed or Refractory Multiple Myeloma.
Authors: Malhotra Binu , Porter David L , Stadtmauer Edward A. , Schuster Stephen J. , Vogl Dan T.
Published: October 2019

Abstract 4942

Background

Patients with relapsed or refractory multiple myeloma (MM) are resistant to most therapies. In this study, we assess the efficacy and tolerability of continuous infusion (CI) cyclophosphamide in a group of heavily pre-treated patients with relapsed/refractory MM.

Methods

Charts of all patients treated with CI cyclophosphamide for relapsed/refractory MM between 01/2003 and 12/2008, at the Hospital of University of Pennsylvania, were identified and reviewed. Patients who had received at least one cycle of CI cyclophosphamide were included for the analysis. The dose of cyclophosphamide ranged between 200-300 mg/m2/day CI for 4 days. Duration of each cycle was planned at 28 days. Response was assessed using the international uniform response criteria for MM. Toxicity was assessed using the National Cancer Institute Common Toxicity Criteria, version 4.

Results

We treated 24 patients with CI cyclophosphamide. The median age was 60.5 years (range 33-75 years) and 66% were male. Patients had received a median of 5 prior regimens (range 2.0 to 11.0) and the median duration of time from diagnosis to treatment with CI cyclophosphamide was 5 yrs (range 0.38-22 yrs). Cyclophosphamide was started in 19 patients for management of progressive disease, and in 5 patients for lack of response or adverse effects from prior therapies. Prior therapies included: bortezomib (in all patients), lenalidomide (in 96% of patients) and autologous stem cell transplant (ASCT - once in 46%, and twice in 29%) among others. The median number of cyclophosphamide cycles administered until the time of last follow up was 4.0 (range 1.0-43.0). Treatment was ongoing in 4 patients at the time of last follow up, and two patients with stable disease were being observed without therapy. Median overall survival for all patients was 22.3 months (95% CI 10.04-34.53) and median progression free survival (PFS) was 7.4 months (95%CI 4.12-10.69). Partial response was noted in 4 patients, who had a median duration of response of 4.2 months and median time to best response of 0.8 month. Disease was stabilized in 14 additional patients. Disease progressed despite therapy in 6 patients, and in 4 of those progressive disease was evident from cycle one. Cytopenias were noted in 8 patients, with 4 related to the treatment with cyclophosphamide, but in only one case did this lead to discontinuation of therapy.

Conclusion

In our experience with 24 patients, monthly cycles of continuous infusion cyclophosphamide was a safe and effective method of treating relapsed or refractory multiple myeloma resulting in a prolonged progression free survival in very heavily pretreated patients.

Disclosures

No relevant conflicts of interest to declare.