bluuebunny/crossref_metadata_2025 huggingface dataset:
Daunorubicin -TiO2 Nanocomposites As ‘smart' pH-Responsive Drug Delivery System,
Authors: Zhang Hai-jun , Chen Bao-An
Published: October 2019

Abstract 3479

Daunorubicin (DNR) with a broad spectrum of anti-tumor activity is limited due to the serious side-effects in the clinical application. The aim of this study was to explore the novel pH-responsive drug delivery system (DDS) based on titanium dioxide (TiO2) nanoparticles (Nps) for its potential roles to enable more intelligently controlled release, enhance chemotherapeutic efficiency, and reduce the side-effects of DNR. DNR was loaded onto the TiO2 Nps by forming (six-membered chelate) complexes with transition metal Ti to contract DNR-TiO2 nanocomposites as DDS. The encapsulation efficiency and loading efficiency of DNR loaded TiO2 Nps were assessed and calculated as 65.46±6.82% and 20.63±3.55%, respectively.The DNR was released from the DDS much more rapidly at pH 5.0 and 6.0 than at pH 7.4. The release behavior is a desirable characteristic for tumor-targeted drug delivery. Most DNR will remain in the carrier for a considerable time period at normal physiological conditions (pH 7.4), indicating the potential for the prolonged DNR retention time in the blood circulation and thereby greatly reducing the side effects to the normal tissues. On the other hand, once the DNR loaded TiO2 Nps are taken up by tumor cells via endocytotic process, a faster release may occur at lower local pH, i.e, inside the endosome and lysosome of cancer cells ((pH 4.5∼6.5), leading to the significant improvement in cancer treatment efficacy. The DNR- TiO2 nanocomposites as DDS induced the remarkable improvement in the anti-tumor activity, which were demonstrated by the flow cytometry, MTT assay and nuclear DAPI staining. Furthermore, the possible signaling pathway was explored by Western blot. For instance, in human leukemia cells (K562 cells), our observations demonstrated that the DDS could obviously increase the intracellular concentration of DNR and enhance its potential anti-tumor efficiency through inducing apoptosis in a caspase-dependent manner, indicating that DNR-TiO2 nanocomposites could act as an efficient DDS importing DNR into target cancer cells. These findings revealed that such ‘smart' DNR delivery strategy represent a promising approach in hematologic malignancy therapy.

Disclosures:

No relevant conflicts of interest to declare.
Combining Tetrahydrouridine with Decitabine Addresses Malignant Cell Sanctuary in the Liver, a Tissue That Expresses High Levels of Cytidine Deaminase,
Authors: Ebrahem Quteba , Mahfouz Reda Z , Peng Kwok Ng , Saunthararajah Yogen
Published: October 2019

Abstract 3509

Non-cytotoxic DNA methyl-transferase 1 (DNMT1) depletion by decitabine induces p53-independent differentiation mediated cell cycle exit in acute myeloid leukemia (AML) cells and spares normal hematopoietic stem cells, providing a potentially important alternative to conventional apoptosis-based therapy. However, there are pharmacologic barriers that impede optimal clinical translation of pre-clinical observations. One major barrier is the enzyme cytidine deaminase (CDA), which drastically reduces the half-life of decitabine from many hours in vitro to a few minutes in vivo. This problem is significant since decitabine is S-phase specific in its mechanism of action, and for effective DNMT1 depletion, low peak levels but extended half-life are optimal. Furthermore, CDA is highly expressed in the liver and intestines, generating potential sanctuary sites from the effects of decitabine. Finally, upregulation of CDA expression is a potential mechanism by which AML cells may resist decitabine at the cellular level. To overcome the multiple CDA-mediated barriers to effective therapy, we evaluated the combination of very low dose decitabine with the CDA inhibitor tetrahydrouridine (THU) in a murine xenotransplantation model of aggressive human AML (THP1 AML cells – p53 and p16-null). Treatment groups (n=5 per group) received equal volume vehicle (PBS), low dose cytarabine (0.1 mg/kg subcutaneous [SC]) with (THU) (4mg/kg intraperitoneal [IP]), decitabine (0.1 mg/kg SC) with THU (4mg/kg IP), and decitabine alone (0.2 mg/kg SC). Median survival in decitabine (61 days) and THU-decitabine (70 days) treated mice was significantly extended compared to the PBS (38 days) and THU-cytarabine (50 days) treatment groups (Log Rank p=0.00156 (figure 1). In decitabine treated mice, eventual emergence of disease was concentrated in the liver, consistent with a sanctuary function of this organ. Combination of THU with decitabine largely eliminated liver infiltration by AML, reducing liver tumor load from average 5.2g in PBS treated mice to 0.8g in THU-Decitabine treated mice (t-test p<0.001, livers were harvested at different time-points corresponding to the Kaplan-Meier curve). In separate experiments without AML cell xenotransplantation, the non-cytotoxic, DNMT1 depleting character of the decitabine therapy was confirmed by hematologic and bone marrow evaluation for DNA damage by phospho-H2AX staining. In conclusion, combining THU with decitabine can address malignant cell sanctuary in the liver, an organ that expresses high levels of cytidine deaminase. The ability of this combination to address other aspects of CDA-mediated resistance to therapy is also being evaluated.

Disclosures:

No relevant conflicts of interest to declare.
AML Depends on VEGFR1 and Not VEGFR2 for Proliferation and Protection From Chemotherapy,
Authors: Bais Swarna , Wise Elizabeth , Harris Eric M. , Meacham Amy , Cogle Christopher R
Published: October 2019

Abstract 3598

Background:

Vascular endothelial growth factor (VEGF) and its cognate receptors (VEGFRs) are known to serve important roles in normal hematopoiesis, but the importance of the VEGF/VEGFR axis in malignant hematopoiesis, namely acute myeloid leukemia (AML), is poorly defined.

Methods:

Bone marrow from AML patients were analyzed for VEGFRs on the malignant myeloblast populations. Human myeloid leukemia cell lines, KG-1, HL60 and K562, were also evaluated for VEGFR expression. Knockdown of VEGFR1 and VEGFR2 receptors in AML cells expressing these receptors were performed using lentivirus transfection of shRNA. Cell proliferation was quantified using XTT colorimetric assay and apoptosis was evaluated by Annexin V and PI staining.

Results:

Bone marrow from 12 consecutive AML patients showed that 33% of patients had malignant myeloblasts that expressed VEGFR1. Of the AML cell lines tested, only KG-1 cells expressed VEGFR1 and VEGFR2. Knockdown of VEGFR1 and VEGFR2 in KG-1 cells were confirmed by Western blot. When VEGFR1 was knocked down in KG-1 leukemia cells, cell proliferation was significant decreased (Figure 1). In addition, knocking down VEGFR1 resulted in enhanced sensitivity to cytarabine chemotherapy (Figure 1). In contrast, knockdown of VEGFR2 did not result in changes in leukemia cell proliferation or sensitivity to cytarabine chemotherapy (Figure 2). When KG-1 leukemia cells knocked down for VEGFR1 expression were transplanted into sublethally, irradiated NOD/scid/IL2R□−/− (NSG) mice (n=10), none of the mice showed engraftment of VEFR1 deficient cells.

Conclusions:

The VEGF/VEGFR axis is an important determinant in AML pathobiology. In specific, AML cells depend upon VEGFR1 and this receptor represents a promising target for future therapeutic intervention.

Disclosures:

No relevant conflicts of interest to declare.
Synergistic Depression of Wnt Pathway by Sequential Combination of Decitabine and Idarubicin in Human Myeloid Leukemic Cell Lines,
Authors: Tong Hongyan , Li Kongfei , Mei Chen , Jin Jie
Published: October 2019

Abstract 3628

PURPOSE:

In the preliminary experiment, we investigated the effect of anti-leukemia drugs (idarubicin, daunorubicin cytarabine, thalidomide, homoharringtonine) in combination with decitabine of inhibiting growth of myeloid leukemic cell lines. The result showed notable synergistic effect of sequential combination of decitabine and idarubicin. In searching the associated mechanism, we assessed the effect of sequential combination of decitabine and idarubicin in apoptosis, cell cycle arrest, and depression of wnt pathway. Methods: Myeloid leukemia cells (U937, HEL and SKM-1) were treated with decitabine alone, idarubicin alone, and decitabine sequential with idarubicin to determine their impact on cellular proliferation, cell cycle regulation, apoptosis and the regulation of wnt pathway by detecting DNA methylation status, mRNA level and protein expression. Results: The sequential combination of decitabine and idarubicin produced synergistic inhibition of myeloid leukemia cells growth by apoptosis and cell cycle arrest. Sequential treatment with both agents induced synergistic effect on decreasing methylation status of wnt antagonists (SFRP1, HDPR1 and DKK3) which result in re-expression or up-expression of these genes. Furthermore, we found that the down-steam genes of wnt pathway including c-myc and β-catenin were down-regulated. Conclusion: Taken together, these findings showed that decitabine sequentially combined with idarubicin has synergistic anti-leukemia effect, which suggest clinical potential in the treatment of myeloid leukemias.

Disclosures:

No relevant conflicts of interest to declare.
The Functional Role of TLR9 in Human Platelets
Authors: Thon Jonathan Noah , Peters Christian G. , Aslam Rukhsana , Rowley Jesse , Weyrich Andrew S. , Semple John W. , Flaumenhaft Robert C. , Italiano Joseph E.
Published: October 2019

Abstract 366

Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. This is perhaps best exemplified by the observation that severe thrombocytopenia is associated with sepsis. While recent phenotypic and functional studies have addressed the roles of TLRs 2 and 4 in human/murine PLTs, which are primarily extracellular receptors, very little is known about the cellular localization, expression, and physiological significance of intracellular TLR9. Here we show that TLR9 localizes to unique granular compartments along the cell periphery (underlying the plasma membrane) in human PLTs. While thought to reside exclusively in the endosomes of monocytes, macrophages, and plasmacytoid dendritic cells, TLR9 expression is significantly enhanced in PLTs following incubation with thombin, ADP, PMA, CRP and type IV collagen, suggesting activation-mediated granule release. Surprisingly, TLR9 surface expression did not coincide with CD62P and CD61 expression levels upon PLT activation (which were not increased following type IV collagen incubation), and TLR9 was shown not to co-localize with known alpha-granule (fibrinogen, CD62P, PDGF-B, VEGF, CD42a, CD42b), dense granule (serotonin), endosomal (M6P, syntaxin-13), or lysosomal (LAMP-1) proteins. Immunogold electron microscopy revealed that TLR9 was expressed instead in a novel intracellular electron-dense granule (T-granule) that is mostly distributed along the plasma membrane. Incubation of resting PLTs with synthetic unmethylated Type C CpG dinucleotides (characteristic of bacterial/viral DNA) resulted in increased TLR9 surface expression, followed by increased Type C CpG sequestration and CD62P surface expression over 20 minutes. Interestingly, when TLR9 surface expression was specifically upregulated by pre-incubating PLTs with type IV collagen, subsequent incubation with Type C CpG resulted in considerably increased Type C CpG sequestration, CD62P surface expression, and PLT aggregation within 30 seconds of addition. These results imply that PLTs must be primed to express TLR9 on their surface prior to signal transduction through TLR9, and provide a mechanism for PLT regulation of the immune response to infection in human whole blood by sequestering bacterial/viral DNA and marking themselves for clearance. Taken together, this paper; (1) tracks TLR9 to a new intracellular compartment in PLTs, (2) describes a novel mechanism of TLR9 signaling, and (3) reveals a unique role for human PLTs as mediators of innate immunity at sites of vascular damage.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation in the Context of Daily Practice of Follicular Lymphoma patients' Outcome Before and After Approval of Rituximab in First Line Therapy in This Indication: Results of a Retrospective Analysis of 247 Unselected Patients with a 5-Year Median of Follow-up,
Authors: Virelizier Emmanuelle Nicolas , Segura-Ferlay Celine , Domnisoru Izabela-Irina , Rey Philippe , Gargi Thérèse , Chassagne-Clément Catherine , Favier Bertrand , Biron Pierre , Belhabri Amine , Sebban Catherine , Blay Jean-Yves , Ghesquieres Herve
Published: October 2019

Abstract 3698

Background:

Randomized trials had demonstrated that rituximab improved outcome in newly diagnosed follicular lymphoma (FL) and this anti-CD20 monoclonal antibody was approved in France since 2004 in combination of chemotherapy (CT) for LF in first line therapy. As these results were obtained in selected patients with strict inclusion and exclusion criteria used in clinical trials, whether the improvement of patient's outcome is valuable in unselected population of FL patients with various medical history, different CT regimens is questionable. We performed a retrospective analysis to evaluate if the management and the outcome of FL have significantly changed in our institution before and after rituximab approval in 2004.

Patients and Methods: all adult patients referred for first line LF were included and separated into cohort 1 diagnosis between 1996 and 2003 (103 patients) and cohort 2 between 2004 and 2010 (144 patients). Baseline clinical and biological datas, Follicular Lymphoma International prognostic Index (FLIPI), type of therapy, treatment response, progression free survival (PFS) and overall survival (OS) were compared.

Results:

There were no statistical differences in patients' characteristics between the 2 cohorts, including FLIPI score. In cohort 2, 71% received in first line therapy a rituximab based regimen (19% in monotherapy and 52% with CT). In cohort 1, 58% of patients were treated with CT and 10% with rituximab, 2% in monotherapy and 8% in combination with CT. Response rate were significantly improved in cohort 2 compared to cohort 1: complete response rate was 74.3% versus 57.8%, P <.001. With a median follow-up of 115.5 and 46.5 months in cohort 1 and 2, the 2-year PFS was 67% for cohort 1 and 82% for cohort 2 (P =.0039), respectively. The 2-year OS was also improved between the two periods (98% versus 87%, P =.0007). We could confirm the prognostic value on OS of the FLIPI score in whole series, in cohort 1 before the rituximab era but not in cohort 2. In fact, the 2-year OS was respectively 100% in low risk, 98% in intermediate risk and 95% in high risk (P =.14)/

Conclusions:

These datas confirms the improvement of FL patients' outcome observed in clinical trials after the approval of rituximab in first line therapy prescript in a context of daily practice whatever age, medical history, and type of CT.

Disclosures:

No relevant conflicts of interest to declare.
Rituximab Followed by Involved Fields Radiotherapy (IF-RT) in Stage I-II Follicular Lymphoma (FL): Long Term Results,
Authors: Ruella Marco , Filippi Andrea , Russo Anna DI , Caracciolo Daniele , Matteucci Paola , Magni Michele , Di Nicola Massimo , Ricardi Umberto , Montefusco Vittorio , Parvis Guido , Tarella Corrado , Gianni Alessandro M. , Devizzi Liliana
Published: October 2019

Abstract 3699

Background:

Radiotherapy is considered the standard approach in stage I-II non bulky FL but no consensus of the required radiation field has been reached and failure-free survival (FFS) reported ranged between 41 to 49%. Published randomized trials failed to demonstrate the superiority of non adriamycin-containing regimens plus RT over RT alone and only one single arm study with COP-B plus IF-RT reported superior FFS (73%) however complicated by an high rate of treatment related toxicity. Since the monoclonal anti CD-20 antibody Rituximab has shown to be effective in systemic therapy of FL as primary treatment as well at relapse, the combination of the anti-CD20 Rituximab with IF-RT may be considered a valid approach, allowing an effective systemic disease control while sparing the toxicity of chemotherapy. We reported here the long-term outcome of a series of stage I-II FL pts treated front-line with Rituximab followed by IF-RT. Patients and Methods: From July 1999 to April 2009, 36 consecutive stage I-II FL WHO grade I-II, have been enrolled in this phase II study and treated with 4 weekly doses of Rituximab followed by IF-RT. Median age: 49 yrs (range 34–82), M/F: 20/16; FLIPI 0–1: 36; Bulky:0. BM PCR analysis to detect minimal residual disease (MRD) was carried out in 25 pts with a molecular probe: PCR+/PCR-: 10/15. Treatment consisted of 4 weekly doses of Rituximab (375 mg/sqm) followed by external bean RT on involved fields. Median radiation dose was 30.6 Gy, (range 20–40). Results: Rituximab followed by IF-RT was well tolerated without any additional toxicity. All but one pts reached a complete remission (CR). With a median follow-up of 7 years (range: 2–12), 34 pts are alive and 24 in continuous complete remission. Of the 12 relapsed pts, 6 were PCR+ versus 4 PCR- at baseline BM analysis; 2 pts had no molecular probe. Two smokers pts died of lung cancer at 3 and 4 years after immuno-radiotherapy. The OS and FFS were 94% and 67% respectively. These results compare favorably with those observed in similar historical control group treated at our Institution with IF-RT alone (OS:87%,FFS:46.8%). Conclusion: Rituximab followed by IF-RT is a well tolerated and effective front-line treatment for limited stage FL. Long-term results are comparable to those reported for non-anthracycline-containing chemo-radiotherapy and higher when compared to radiotherapy alone, making Rituximab-IF-RT a potential option as initial therapy of limited stage FL.

Disclosures:

No relevant conflicts of interest to declare.
Cycling Toward Leukemia Stem Cell Elimination Wtih a Selective Sonic Hedgehog Antagonist,
Authors: Shih Alice Y , Schairer Annelie , Barrett Christian L , Geron Ifat , Recart Angela C Court , Goff Daniel , Prashad Sacha , Wu Jerry , Jiang Qingfei , Gotlib Jason , Balaian Larisa , Minden Mark D. , Leu Heather , Wall Russell , Ma Wenxue , Shazand Kamran , McPherson John D , Kornblau Steven M. , Deichaite Ida , Pu Minya , Bao Lei , Martinelli Giovanni , Reya Tannishtha , Morris Sheldon R , VanArsdale Todd , Hudson Thomas J , Messer Karen , Mikkola Hanna , Levin Wendy J. , Frazer Kelly A , Sadarangani Anil , Jamieson Catriona
Published: October 2019

Abstract 3776

Cumulative evidence suggests that dormant self-renewing leukemia stem cells (LSC) contribute to relapse and blast crisis transformation by evading therapies that target cycling cells. Previously, sonic hedgehog (Shh) signaling was shown to modulate cell cycle regulation and self-renewal in normal mouse hematopoietic stem cells. However, its role in human LSC regeneration and quiescence had not been elucidated. Here we investigated the role of Shh signaling in maintenance of dormancy. We show that, compared to chronic phase CML and normal progenitors, human blast crisis LSC harbor enhanced expression of the Shh transcriptional activator, GLI2, and decreased expression of a transcriptional repressor, GLI3. Treatment of human blast crisis LSC engrafted RAG2−/−gc−/− mice with a selective Shh inhibitor, PF-04449913, reduced leukemic burden in a niche-dependent manner commensurate with GLI downregulation. Full transcriptome RNA sequencing performed on FACS-purified human progenitors from PF-04449913 treated blast crisis LSC engrafted mice demonstrated greater Shh gene splice isoform concordance with normal progenitors than vehicle treated controls. In addition, RNA sequencing revealed significantly decreased cell cycle regulatory genes expression and splice isoform analysis demonstrated reversion towards a normal splice isoform signature for many cell cycle regulatory genes. Moreover, cell cycle FACS analysis showed that selective Shh inhibition permitted dormant blast crisis LSC to enter the cell cycle while normal progenitor cell cycle status was unaffected. Finally, PF-04449913 synergized with BCR-ABL inhibition to reduce blast crisis LSC survival and self-renewal in concert with increased expression of Shh pathway regulators. Our findings suggest that selective Shh antagonism induces cycling of dormant human blast crisis LSC, rendering them susceptible to BCR-ABL inhibition, while sparing normal progenitors. Implementation of novel LSC splice isoform detection platforms to assess efficacy of Shh inhibitor-mediated sensitization to molecularly targeted therapy may inform dormant cancer stem cell elimination strategies that ultimately avert relapse.

Disclosures:

Levin: Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership.
Heparanase Activation of the Coagulation System in a Mice Sepsis Model
Authors: Henig Israel , Axelman Elena , Brenner Benjamin , Vlodavsky Israel , Nadir Yona
Published: October 2019

Abstract 378

Background:

Heparanase that is abundant in platelets and neutrophils is implicated in cell invasion, tumor metastasis and angiogenesis. Recently, we demonstrated that heparanase is directly involved in the regulation of the hemostatic system. Heparanase was shown to form a complex and enhance tissue factor (TF) activity, resulting in increased factor Xa production (Nadir et al, Haematologica, 2010). In the present study, the effect of heparanase on sepsis was investigated. Methods: ICR mice, heparanase knock-out mice and heparanase over-expression mice were studied. Sepsis was induced by intra-peritoneal injection of lipopolysaccharides (LPS). Four hours after injection, blood was obtained via temple plexus puncture followed by mice sacrificing. Levels of thrombin-antithrombin (TAT), D-Dimer and IL-6 were tested using ELISA. Plasma factor VII levels were evaluated using the Western blot analysis and fibrinogen levels in the lung, kidney and spleen were estimated by immunostaining. Results: Intra-peritoneal injection of heparanase (0.5μg/mg) compared to PBS injection, increased the TAT (p<0.05) and D-Dimer (p<0.05) levels, which were similar to the ones induced by LPS (5μg/mg), although the IL-6 levels did not rise and mice did not show signs of illness. Moreover, heparanase injection half an hour following LPS administration resulted in reduced levels of TAT, D-Dimer, fibrinogen and of factor VII (p<0.001), increased levels of IL-1 (p<0.001) and profuse blood leak from puncture, compatible with severe disseminated intravascular coagulation (DIC). Similarly, injection of LPS to heparanase knock-out mice led to lower levels of TAT (p<0.001), D-Dimer (p<0.001) and IL-6 (p<0.001) compared to control mice, while injection of LPS to heparanase over-expression mice resulted in DIC. Conclusions: Heparanase is found to induce activation of the coagulation system without inflammatory response. It also contributes to laboratory enhancement of sepsis resulting in DIC. Inhibitors of heparanase may potentially attenuate morbidity and mortality in sepsis.

Disclosures:

No relevant conflicts of interest to declare.
Outcome of Complex Variant Philadelphia Positive Chromosome Translocations in the Era of Tyrosine Kinase Inhibitors,
Authors: Shah Bijal D. , Zhang Ling , Bulkeley William , Moscinski Lynn , Papenhausen Peter , Pinilla-Ibarz Javier
Published: October 2019

Abstract 3788

Background:

It is important to prospectively identify patients with CML who might not respond to conventional tyrosine kinase inhibitory therapy. Cytogenetic variants of the Philadelphia (cvPh) chromosome can be observed in 5–8% of patients diagnosed with Chronic Myelogenous Leukemia (CML), and usually involve at least one or more chromosome other than 9 and 22. Specifically, cvPh refers to patients with (V;9;22), where “V” indicates one or more additional translocation partners. Previous data suggest that patients with cvPh may respond poorly to therapy (Gorusu et al, 2007), however, many were treated prior to routine use of imatinib, nilotinib, or dasatinib.

Methods:

We conducted a retrospective review of 338 patients with CML treated at Moffitt Cancer Center to investigate the presence of cvPh and the outcome of these patients under treatment with TKI therapy.

Results:

Of 338 patients we identified 12 patients with cvPh (3.5%). Demographic data show male to female ratio of 5:7, and median age 50 (35–76). Two were in accelerated phase at the time of diagnosis, the remaining were in chronic phase. Baseline cytogenetics from time of diagnosis were not available in six patients. Median time to development of cvPh was 10 months (0–74). Two did not have clearly identified cvPh at diagnosis but both had complex karyotype. Eleven patients were treated with imatinib, among whom only one has shown a complete cytogenetic response (CCR). Two others continue on imatinib with unknown response status. Four patients were treated with dasatinib, including one in the frontline setting. No CCR's have been documented in this group, with two patients pending reevaluation. Seven patients were treated with nilotinib after failing a prior TKI, of whom only one has shown a CCR, with response evaluation pending in two patients. Two patients have undergone allogeneic stem cell transplant, with resolution of disease, including the nilotinib treated patient in CCR above. In this series of 12 patients, only three have shown resolution of variant translocation with a median followup of 45 months. All patients are alive at the last follow up.

Conclusion:

In this retrospective review, patients with cvPh appear to have a more resistant genomic type, and seems to be a bad prognostic factor for response to TKI therapy.

Disclosures:

No relevant conflicts of interest to declare.
Ponatinib Is Active Against the CUX1-FGFR1 Fusion Kinase and Against Imatinib Resistance Mutations of the FIP1L1-PDGFRα Fusion Kinase and of KIT,
Authors: Lierman Els , Smits Sanne , Cools Jan , Dewaele Barbara , Debiec-Rychter Maria , Vandenberghe Peter
Published: October 2019

Abstract 3848

Imatinib (IM) was initially developed as a small molecule inhibitor of the BCR-ABL1 kinase, but also potently inhibits other oncogenic kinases, such as PDGFRβ and PDGFRα fusion proteins. IM has revolutionized the treatment of chronic myeloid leukemia (CML) and other neoplasms, but the development of IM resistant mutations has emerged as an important problem, triggering a search for novel compounds that overcome resistance. A leading third generation candidate is ponatinib, a novel multikinase inhibitor with potent activity towards BCR-ABL1, KIT, FGFR1, PDGFRα and other kinases. Importantly, ponatinib also targets numerous IM resistant BCR-ABL1 kinase domain mutations including the panresistant T315I mutation.

We investigated the effect of this compound on IM resistant kinase mutations in lymphoid/myeloid neoplasms associated with eosinophilia and rearrangements of PDGFRα and FGFR1, as well as KIT associated malignancies. Ba/F3 cells were used expressing either FIP1L1-PDGFRα, the IM resistant FIP1L1-PDGFRα-T674I mutant, the panresistant FIP1L1-PDGFRα-D842V mutant, or the novel CUX1-FGFR1 fusion. In addition, several KIT mutants were investigated. The growth of FIP1L1-PDGFRα and IM resistant FIP1L1-PDGFRα-T674I mutant expressing cells was strongly inhibited by ponatinib with IC50 values of 0,6 nM and 9 nM respectively. Also the panresistant FIP1L1-PDGFRα-D842V mutant and the novel CUX1-FGFR1 fusion responded well to ponatinib treatment with 50% growth inhibition at 154 nM and 56 nM respectively. IL3-driven growth of Ba/F3 cells was resistant to ponatinib (IC50: 2 μM). Western blot analysis confirmed the direct effect of ponatinib on the auto-phosphorylation of the PDGFRα and FGFR1 fusion proteins, as well as on the downstream signaling protein STAT5. Finally, we investigated several KIT single and double mutants and preliminary data indicate an inhibitory effect of ponatinib towards several KIT mutants.

In conclusion, our results demonstrate the in vitro activity of ponatinib against IM resistant mutants of the FIP1L1-PDGFRα fusion kinase, against the CUX1-FGFR1 fusion kinase as well as against IM resistant KIT mutations. Our data indicate that ponatinib, which is currently under investigation in phase II clinical trials for IM resistant CML, may also be active against neoplasms driven by FGFR1, PDGFR or KIT kinase activity, and able to overcome IM resistance in these malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Metascoring and Gene Expression Profiling in Clinical Routine in Multiple Myeloma,
Authors: Meißner Tobias , Seckinger Anja , Rème Thierry , Hielscher Thomas , Möhler Thomas , Neben Kai , Goldschmidt Hartmut , Klein Bernard , Hose Dirk
Published: October 2019

Abstract 3940

BACKGROUND.

Multiple myeloma is characterized by molecular heterogeneity transmitting to survival ranging from several months to over 15 years. Gene expression profiling allows assessment of biological entities, risk, and targets. Its translation into clinical routine alongside conventional prognostic factors has been prevented by lack of appropriated reporting tools and the integration with other prognostic factors into a single risk stratification (metascoring).

METHODS.

We present here a non-commercial open source software-framework developed in the open source language R (GEP-report) containing a graphic user interphase based on Gtk2. Affymetrix microarray raw-data and a documentation-by-value strategy to directly apply thresholds and grouping-algorithms from a reference cohort of 262 myeloma patients are used. Gene expression-based and conventional prognostic factors are integrated within one risk-stratification (HM-metascore) and the final report is created as a PDF-file.

RESULTS.

The GEP-report comprises i) quality control, ii) test of sample identity, iii) biological classifications of multiple myeloma, iv) risk stratification, v) assessment of target-genes, and vi) integration of expression-based and clinical risk factors within one metascore. This HM-metascore sums over the weighted factors gene-expression based risk-assessment (UAMS-, IFM-score), proliferation, ISS-stage, t(4;14), and expression of prognostic target-genes (AURKA, IGF1R) for which clinical grade inhibitors exist. It delineates three significantly different groups of 13.1, 72.1 and 14.7% of patients with a 6-year survival of 89.3, 60.6 and 18.6%, respectively.

CONCLUSION.

GEP-reporting allows prospective assessment of target gene expression and integration of current prognostic factors within one risk stratification (metascoring), being customizable regarding novel parameters or other cancer entities.

Disclosures:

No relevant conflicts of interest to declare.
Regulatory T Cells of G-CSF Mobilized Stem Cell Donors Qualify for Clinical Applications,
Authors: Ukena Sya N. , Velaga Sarvari , Lilia Goudeva , Ivanyi Philipp , Ganser Arnold , Franzke Anke
Published: October 2019

Abstract 4055

Recent clinical studies demonstrate the high potency of human regulatory T cells (Tregs) to control graft-versus-host disease following hematopoietic stem cell transplantation (SCT). Isolation of Tregs after recombinant G-CSF induced mobilization of stem cells would simplify the design of clinical trials in allogeneic SCT. However, there is growing evidence that G-CSF also exerts profound immune modulatory effects in the adaptive immune system. Therefore, we analyzed Tregs isolated by FACS based cell sorting from stem cell donors before (n=8) and after (n=13) G-CSF administration regarding their phenotype, stability, functional and expansion properties. Absolute CD4+ T cell (3.2 fold increase of mean after G-CSF; p<0.05) and CD4+CD25hi Treg cell numbers (4.1 fold increase of mean after G-CSF; p<0.01) were significantly increased after G-CSF administration. Analysis of the Foxp3 TSDR demethylation level displays a stable Foxp3 phenotype of G-CSF encountered Tregs (mean value 97.1% vs. 95.0 % after G-CSF administration). Moreover, the CD4+CD25hi Tregs of G-CSF treated SC donors suppress the proliferation of effector T cells with no significant differences to Tregs isolated from healthy donors before G-CSF treatment (mean values 42.1% vs. 49.9% after G-CSF administration at a Treg/ T effector cell ratio of 1:1). In vitro expansion of Tregs isolated after G-CSF application with anti-CD3 and anti-CD28 dynabeads in the presence of interleukin-2 led to comparable cell numbers as for the stimulation of control Treg cells. However, differences could be detected for the thymic derived marker molecule CD31 and those associated with activation (LAP, CD69, CD62L) and migration (CxCR3) as detected by FACS analysis. Our results show no significant differences regarding suppressive function and stability of Tregs isolated from stem cell donors before and after G-CSF administration. These data support the application of G-CSF mobilized Tregs for clinical trials which in turn opens up new possibilities for the adoptive transfer of Tregs in HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Effects of Prophylactic Defibrotide for Veno-Occlusive Disease in Hematopoietic Stem Cell Transplantation
Authors: Park Meerim , Park Hyeon Jin , Eom Hyeon-Seok , Park Jeong A , Lim Yeon Jung , Yoon Jong Hyung , Lee Hye Won , Yun Tak , Park Byung-Kiu
Published: October 2019

Abstract 4517

Background:

Defibrotide has recently been successfully used to treat veno-occlusive disease (VOD). We report the use of intravenous defibrotide for VOD prophylaxis in 49 patients who received hematopoietic stem cell transplantation (HSCT). This group was compared with a control group of 49 patients who underwent HSCT without defibrotide prophylaxis.

Patients and methods:

Data were collected retrospectively on 98 patients undergoing HSCT at the National Cancer Center, Goyang, Korea, between August 2005 and July 2008. During this period, defibrotide was randomly supplied to 49 patients free of charge by the compassionate use program. For 49 patients, VOD prophylaxis with 200 to 400 mg of defibrotide (25 mg/kg/day in children weighing less than 30 kg) administered intravenously 3 or 4 times daily was initiated from the start of conditioning and continued until 28 days post-HSCT.

Results:

VOD was diagnosed in 5 of 98 total patients (5.1%) at a median of 11 days post-HSCT (range: 10–14 days). VOD was graded as mild in 2 patients, moderate in 1, and severe in 2. The incidences of engraftment syndrome, thrombotic microangiopathy and VOD were not significantly different between the defibrotide group and the control group, although absolute incidences of each syndrome were lower in the defibrotide group. Patients who received prophylactic defibrotide showed earlier platelet engraftment (p<0.01) and lower activated prothrombin time (p=0.02) after HSCT. There was no day 100 treatment-related mortality (TRM) in the defibrotide group. Day 100 TRM occurred in two patients who did not receive defibrotide for VOD prophylaxis during allogeneic HSCT. Each patient was diagnosed with mild or severe VOD, and the direct causes of death were infection or VOD. Of the patients who were diagnosed with VOD, the overall survival at 100 days post HSCT was significantly higher in the defibrotide group compared to the control group (100% vs. 33.3 ± 27.2%, p<0.01). Defibrotide was well tolerated and was not discontinued in any patients. There was no grade 3 or 4 toxicity related to defibrotide or any worsening of clinical bleeding.

Conclusions:

Our data showed that the use of prophylactic defibrotide is safe and may have beneficial effects of defibrotide on hepatic endothelial damage following HSCT, with no TRM in defibrotide group. Considering there is no consensus on when to start treatment if a patient shows clinical features suggesting VOD, it could be important to use prophylactic defibrotide in advance for improved VOD outcomes.

Disclosures:

No relevant conflicts of interest to declare.
Autologous Stem Cell Transplantation for NHL: 16 Years Follow- up
Authors: Guillermo Cecilia , Lipschutz Judith , Zunino Juan , Topolansky Laura , Rocca Alejandra , Stevenazzi Mariana , Diaz Andrea , Perdomo Susana , Perdomo Ana , Lavagna Graciela M , Isaurralde Hugo , Diaz Lilian , Nese Martha
Published: October 2019

Abstract 4520

Between June 1995 and June 2011, we performed 133 Autologous Stem Cell Transplantation (ASCT) in patients with NHL. Long term results of treatment and outcome were analyzed. The data presented were obtained from the Statistical Center of the Center for International Blood and Marrow Transplant Research. Median age was 48 years (range 18–65), 9% ≥ 60 years, 77 males and 55 females. The histology was: 98 (74%) B NHL, Follicular Lymphoma (FL) 26 (20%), Diffuse large B Cell Lymphoma (DLBCL) 47 (35%), Primary Mediastinal Large B Cell Lymphoma 12 (9%), Mantle cell Lymphoma (MCL) 6(4.5%), Marginal Zone Lymphoma 4 (3%), Small Lymphoplasmocytic 2 (1.5%), precursor B Lymphoplasmoblastic 1 (1%), and 35 (26%) T NHL, Peripheral T Cell Lymphomas NOS 12 (9 %), Anaplastic LC, T/N Cell systemic 9 (6.5%), Anaplastic LC, T/N Cell cutaneous 1(1%) and T/NK Cell Lymphoma 7 (5%), Angioinmunoblastic T Cell Lymphoma 2 (1.5%), Mycosis Fungoides 2 (1.5%), other T Lymphoma 2 (1.5%). We emphasize that 59 % of the patients had advanced disease at the moment of the transplant: 41% CR1, 11% CR2, 5% REL1, 8% primary induction failure, other 33% (CR3+, PR, and REL2+), and 2% missing/unknown. Condition regimens were BEAC 88%, BEAM 10% and CBV 2%. Stem cells source: 74% peripheral blood stem cells (PBSC) and 26% received bone marrow (BM) and PBSC.

Overall survival was 86% (95 CI, 80–92) at 1 year, 78% (95 CI, 70–85) at 3 years and 71% (95 CI, 62–79) at 5 years. Median follow up of survivors was 96 months (3–181). Primary cause of death was relapsed or progression.

We concluded that high dose therapy and Autologous Stem Cell Transplantation is an effective and feasible therapy for patients with high risk NHL. However relapse continues to be the most common cause of treatment failure. Newer strategies such monoclonal antibodody, require further investigation but early reports appear promising in reducing relapses in ASCT.

Disclosures:

No relevant conflicts of interest to declare.
Kinetics of Engraftment and Predictors of Outcome in Patients with Advanced Chronic Lymphocytic Leukaemia (CLL) After Treatment with Low Dose Total Body Irradiation (TBI) Followed by related or unrelated hematopoietic Stem Cell Transplantation
Authors: Hebenstreit Karin , Iacobelli Simona , Eisfeld Ann-Kathrin , Pfrepper Christian , Lange Thoralf , Krahl Rainer , Al-Ali Haifa K. , Becker Cornelia , Pönisch Wolfram , Behre Gerhard , Niederwieser Dietger
Published: October 2019

Abstract 4532

Introduction:

In an unicenter analysis we investigated the impact of allogeneic hematopoietic cell transplantation (RIC-HCT) after reduced intensity conditioning on the kinetics of engraftment and predictor of outcome in 50 patients with advanced CLL.

Patient and Methods:

Patients in advanced stage CLL (n=50) received Fludarabine 30mg/m2 on day -4 to -2 and 2Gy TBI on day 0 followed by cyclosporine and MMF from unrelated (n=40) or related (n=10) donors between June 1999 and March 2010 at the University of Leipzig. The majority of patients (n= 35) were male, had Binet C (n=37, 74%) at RIC-HCT. The median age was 58 (range 44–69) years. Of 48 patients for whom cytogenetic analyses were available, 22 (46%) had unfavourable cytogenetics including del 17 or del 11q. Three (6%) patients had CR, 27 (54%) PR, 7 (14%) progressive disease, 12 (24%) stable disease and one (5%) relapse at RIC-HCT. Resistance to first line therapy was present in 25 (50%) patients, whereas 12 (24%) were resistant to Fludarabine. Richter’s transformation was found in six patients (12%). Chimerism was detected in bone marrow and peripheral blood on T-lymphocytes and B-lymphocyte subpopulation after sorting at monthly intervals in the posttransplant period and than in 6 months interval.

Results:

Hematological toxicities after RIC-HCT were moderate. The majority of patients (96%) engrafted with neutrophiles >500/μ L median at day 22 after HCT. Six (12%) and 15 (30%) patients maintained absolute neutrophil counts (ANC) >0,5 × 109/L and platelet counts >50 × 109/L, respectively. T-cell donor chimerism increased to >95% at day 56 and B-cell donor chimerism to 94% at day +360, respectively. B-CLL cells disappeared completely on day +360 (median 0%). Overall survival (OS) at 4 years was 51%, Non relapse related mortality (NRM) 30%, Progression-free survival 33% and progression/relapse 37%. The most common causes of NRM were GvHD (n= 7; 14%) and sepsis (n=3, 6%). Factors significantly associated with increased risk of relapse/progression were intermediate/advanced disease vs. CR/PR1 (p=0.022) and lymphocytes ≥ 5 × 109/L vs. < 5 × 109/L (18% vs. 58%, p= 0.00) at 12 months in univariate analysis.

Conclusion:

Full donor T-cell chimerism was reached early after HCT, while B-cell reconstitution was observed only 1.5 years after RIC-HCT despite the absence of evident disease by 360 days after RIC-HCT. Best predictor for Progression-free survival (PFS) was CR or PR1.

Disclosures:

Pönisch: Mundipharma: Honoraria, Research Funding.
Increased Production of the Inflammatory Cytokine IL12p70 by Activated Human Blood Dendritic Cells Is Associated with Increased Severity of Acute Graft Vs Host Disease Post Allogeneic Hemopoietic Cell Transplantation
Authors: Sartor Mary M , Lau Jenny , Gottlieb David , Bradstock Kenneth F
Published: October 2019

Abstract 4539

Introduction:

Dendritic cells (DC) are centrally involved in the initiation of acute graft versus host disease (GVHD) after allogeneic hemopoietic cell transplantation (alloHCT). We have shown that the activation status of peripheral blood CD11c+ myeloid DC, as assessed by CMRF-44 antigen expression, is highly associated with the occurrence and severity of acute GvHD (Transplantation 2007;83: 839–846). However, very little is known about the function of DC after human alloHCT. We examined the relationship between DC functional properties and the severity of acute GvHD.

Methods:

Peripheral blood CD11c+ myeloid DC from 12 patients were studied weekly up to 8 weeks post transplant for production of IL2, IL4, INFg, IL10 and IL12 using an intracellular cytokine flow assay. Mixed lymphocyte reactions using flow sorted patient DC and third party T cells were used to assess allogeneic immune responses induced by recipient DC in 5 patients.

Results:

IL12 was the only cytokine detected in post transplant DC. Five of 12 patients developed grade II-IV GvHD, the remaining 7 patients developed either no aGvHD or only grade I. In comparison with pre-transplant levels of expression of IL12, patients with grade II-IV GVHD had a significantly higher percentage of CD11c+ DC expressing IL12 (median 15.1%, range 11.2–20.9%) as compared to patients with grade 0-I GvHD in whom there was no change from baseline values (median 6.6% range 2.8–8.9%) p=0.0025. Increased expression of IL12 was observed in CD11c+ DC commencing at day 25 post transplant. Interestingly, analysis of 5 paired samples comparing sorted DC from normal donors with sorted DC from peripheral blood at day +30 post-transplant showed a marked reduction (measured by thymidine uptake from 60% to 90%) in the capacity of post-transplant donor DCs to stimulate 3rd party lymphocyte proliferation. None of these patients developed clinically significant aGvHD.

Conclusion:

Production of IL-12p70 by CD11c+ myeloid DC correlates with severity of GvHD despite impaired capacity of these cells to elicit third party proliferative responses.

Disclosures:

No relevant conflicts of interest to declare.
A Combined Evaluation of Severity, Presentation and Duration of Chronic GvHD Predicts Transplant Outcome Among Patients with a Hematological Malignancy Undergoing Allogeneic Stem Cell Transplantation
Authors: Crocchiolo Roberto , Saillard Colombe , Furst Sabine , El-Cheikh Jean , Castagna Luca , Faucher Catherine , Oudin Claire , Harbi Samia , Granata Angela , Chabannon Christian , Blaise Didier
Published: October 2019

Abstract 4549

Introduction:

chronic graft-versus-host disease (cGvHD) after allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies is known to be associated with lower relapse risk, due to graft-versus-tumor effect. On the other hand, severe, extensive form of cGvHD is associated with high transplant-related mortality (TRM), mainly due to infectious complications as a consequence of the immunosuppressive treatment (IS) of cGvHD. Not only severity of cGvHD but also its presentation and duration of IS is responsible for deleterious effects on transplanted patients.

Here we built and evaluated a score taking into account both severity and duration of cGvHD (and related IS), looking for any association with TRM, OS and relapse/progression after HSCT.

Patients and Methods:

study population is represented by adult patients who received HSCT after fludarabine-busulfan-ATG conditioning between May 1999 and June 2010 at out Institution for a hematological malignancy, and who developed cGvHD. cGvHD was retrospectively classified according to NIH criteria into mild, moderate and severe form. Informations on cGvHD presentation (progressive, quiescent or de novo) were also collected. Duration of IS was taken into account as well as any change in cGvHD severity overtime. A combined mathematical function D was built, taking into account severity, type of presentation and duration of IS (appendix 1), and was treated as a covariate in univariate and multivariate Cox models for TRM, OS and relapse/progression.

Results:

on 313 transplanted patients transplanted in the above mentioned period, 130 developed cGvHD and represented our study population. The function D showed to predict TRM and OS both in univariate and multivariate analysis (table 1); a borderline association was found with relapse/progression, in univariate analysis.

Conclusion:

a function taking into account not only severity but also presentation of cGvHD and duration of IS seems to be predictive of TRM and OS after HSCT. Although further validation is needed, the analytical approach we used here allows to better describe the complexity of post-transplant reality, where severity and duration of IS can change overtime and are associated with patients' outcome.

Disclosures:

No relevant conflicts of interest to declare.