FLT3 Ligand Levels Research Articles

Mouse Modeling of GATA2 Related MDS/AML

GATA2 is a zinc finger transcription factor essential for embryonic and definitive hematopoiesis as well as lymphatic angiogenesis. Human GATA2 deficiency disease is caused by a variety of mutations in GATA2 which lead to the clinical phenotypes variously known as monocytopenia and mycobacterial disease (MonoMac), familial MDS/AML, dendritic cell, myeloid and NK cell lymphopenia (DCML), Emberger syndrome, and classical NK cell deficiency. Patients with GATA2 deficiency have an immunodeficiency with monocytopenia, NK, and B cell leukopenia; are susceptible to mycobacterial, viral and fungal infections as well as MDS and progression to AML. Additionally, patients may suffer from lymphedema and pulmonary alveolar proteinosis. GATA2 mutations have also been associated with aplastic anemia and severe congenital neutropenia.In order to understand the mechanisms underlying the development of MDS/AML in GATA2 deficiency we developed and analyzed a unique mouse model. The previously characterized Gata2+9.5+/- mouse has an intronic regulatory region mutation identical to one found in some patients. However, these mice do not develop MDS/AML spontaneously. We hypothesized that crossing these mice to other MDS/AML prone strains would accelerate the progression of MDS/AML. Specific HOX genes are implicated in this disease process. GATA2-deficient patients often develop monosomy 7 and ASXL1 mutations, both of which lead to the overexpression of HOX genes including HOXA9. The known AML translocation t(2;11)(q31;p15) results in the fusion of NUP98 and HOXD13 (NHD13) and the upregulation of HOX gene expression. Gata2 was identified in a mouse retroviral insertion screen as a potential collaborator in NHD13-mediated leukemogenesis. We hypothesized that hematopoietic specific Hox gene overexpression in a mouse with germline Gata2 haploinsufficiency would lead to the development of MDS/AML. This was done using the established NHD13 transgenic strain crossed to the Gata2+9.5+/- strain. The NHD13 mice express the fusion protein under the control of the hematopoietic specific vav promoter, and have up-regulation of Hoxa5, Hoxa7, Hoxa9, and Hoxa10. Secondly, elevated Flt3 ligand levels have been detected in GATA2-deficient patients as they progress from normal bone marrow morphology to MDS/AML. To model the hyper-activation of the receptor Flt3 by Flt3 ligand, we used a model of constitutively active Flt3, the Flt3-ITD mouse.Gata2+9.5+/- mice were bred to the NHD13 and the Flt3-ITD transgenic strains and compound heterozygotes were analyzed. Gata2+9.5+/-;NHD13 compound heterozygous mice were born normally and at Mendelian ratios. No significant differences in blood cell counts were noted in Gata2+9.5+/-;NHD13 compound heterozygotes until disease progression. These mice developed aggressive AML at 9-14 months of age, similar ages and rates as the NHD13 only mice. Whereas compound Gata2+9.5+/-;Flt3-ITD mice are also born in Mendelian ratios, starting at 10 weeks of age they developed B-cell lymphopenia, similar to that which is seen in human GATA2 deficiency patients displaying B-cell loss early in disease. The Gata 2+9.5+/-; Flt3-ITD mice exhibited increased circulating c-Kit-positive cells in the peripheral blood. Studies to further characterize hematopoiesis in the Gata2+9.5+/-; Flt3-ITD mice, and analyze for the development of MDS/AML are in progress. The Gata 2+9.5+/-; Flt3-ITD mouse model holds promise for the further study of MDS/AML in the context of GATA2 haploinsufficiency. DisclosuresAplan:NIH Office of Technology Transfer: Patents & Royalties.

Potential role of FLT3-ligand in the angiogenic process of multiple myeloma

The aim of the study was to evaluate serum levels of FLT3-ligand (FLT3-L), a soluble molecule in bone marrow (BM), participating actively in hematopoiesis, in relation with angiogenic factors in multiple myeloma (MM) patients. We measured, in 70 patients with active MM and in 38 of them who responded to conventional therapy, serum levels of FLT3-L, along with known angiogenic factors, such as VEGF, endoglin, TNF-alpha and HGF (with ELISA) and BM microvascular density (MVD), estimating the immunohistochemical expression of CD31. All pre-treatment values were higher in active MM patients compared to controls (p<0.001 for all cases), in parallel with both International Staging System and Durie–Salmon stages (p<0.001 for all cases). Moreover, levels of FLT3-L correlated positively with all soluble angiogenic factors, as well with MVD (p<0.0001 for all cases). Post-treatment values of FLT3-L decreased significantly in responders to therapy (p<0.001). The underlying relation of MM angiogenesis with FLT3-L may result from the fact that BM microvasculature is a major source of FLT3-L, both in BM niche and probably in peripheral blood. Our results suggest that serum levels of FLT3-L may be used as angiogenic marker in MM patients.

Serum concentrations of Flt-3 ligand in rheumatic diseases.

Fms-like tyrosine kinase 3 (Flt-3) is a cytokine receptor expressed on the surface of bone-marrow progenitor of hematopoietic cells. Flt-3 ligands are produced by peripheral blood mononuclear cells, and found in various human body fluids. Flt-3 signal is involved in the regulation of vessel formation as well as B cell differentiation, suggesting that Flt-3 signal contributes to the pathogenesis of vascular abnormalities and immune dysregulation in rheumatic diseases. The aim of the present study is to examine serum Flt-3 ligand levels in patients with various rheumatic diseases, and to evaluate the possibility that serum Flt-3 ligand levels can be a useful disease marker. Sera were obtained from 20 dermatomyositis (DM) patients, 36 systemic sclerosis (SSc) patients, 10 systemic lupus erythematosus (SLE) patients, 10 scleroderma spectrum disorder (SSD) patients, 4 mixed connective tissue disease (MCTD) patients, and 12 normal subjects. Flt-3 ligand levels were determined with ELISA. Serum Flt-3 ligand levels were significantly elevated in patients with DM, SSc, SSD and MCTD compared to those in normal subjects. DM patients with elevated Flt-3 ligand levels were accompanied with significantly increased CRP levels and increased frequency of heliotrope rash than those with normal levels. In addition, SSc patients with elevated Flt-3 ligand levels showed significantly reduced frequency of nailfold bleeding. Serum Flt-3 ligand levels can be a marker of cutaneous manifestation in DM and a marker of microangiopathy in SSc. Clarifying the role of Flt-3 ligand in rheumatic diseases may lead to further understanding of these diseases and new therapeutic approaches.

Phase I Trial of Maintenance Sorafenib after Allogeneic Hematopoietic Stem Cell Transplantation for Patients with FLT3-ITD AML

Introduction: The FLT3-ITD mutation is associated with a high relapse rate for patients with AML even after allogeneic hematopoietic stem cell transplantation (HSCT). Sorafenib is an oral tyrosine kinase inhibitor which inhibits the FLT3 tyrosine kinase. Evidence based on FLT3-ligand levels has suggested that the maintenance setting may be the optimal time to administer FLT3 inhibition.Methods: We conducted a phase I trial of maintenance sorafenib after HSCT in patients with FLT3-ITD AML. Patients received a variety of conditioning regimens (12 myeloablative, 10 reduced intensity) and graft sources (19 matched related / unrelated, 1 haploidentical, 1 double umbilical cord blood, 1 mismatched unrelated). All patients were in morphological remission with predominant donor chimerism after HSCT before starting sorafenib. A dose escalation 3+3 cohort design was used to define the maximum tolerated dose (MTD) with an additional 10 patients treated at the MTD. Sorafenib was initiated between days 45 and 120 after HSCT and given continuously for twelve 28-day cycles. The DLT period was the first cycle of treatment.Results: Twenty-two patients with FLT3-ITD AML were enrolled (status at HSCT: CR1=16, CR2=3, refractory=3). The MTD was established at 400 mg BID with one DLT observed (pericardial effusion) which was later deemed unrelated to sorafenib. Two patients died of transplant-related causes and neither was deemed related to sorafenib (idiopathic pneumonia syndrome, cirrhosis from iron overload). Two patients stopped sorafenib after relapse and 5 stopped sorafenib due to attributable toxicities after the DLT period. The most common toxicities observed were skin and GI with dose reductions and interruptions common. There were no significant flares of acute GVHD observed after starting sorafenib and 1-year cumulative incidence of chronic GVHD was 42% (90% CI, 23%, 60%). Effective inhibition of FLT3 as measured by a plasma inhibitory assay was observed at all doses employed. Serial levels of FLT3 ligand were measure in 7 patients. Median level at baseline and prior to any sorafenib administration was 125 pg/ml (range 40-323) and this significantly increased to a median level of 254 pg/ml (range 80-500) (p = 0.016) measured on day 29 of cycle 1.The median follow-up for survivors is 14.5 months (range, 6.6-34.0) after HSCT. One-year progression-free survival is 84% (90% CI, 63%-94%) and one-year overall survival is 95% (90% CI, 79%-99%) (Figure 1). Only one patient who underwent HSCT in CR (n=19) has relapsed.Conclusion: Sorafenib is safe to give as maintenance therapy after HSCT for patients with FLT3-ITD AML and may reduce the rate of relapse. Maintenance sorafenib or other FLT3 inhibitors after HSCT merits further investigation. [Display omitted] DisclosuresChen:Bayer Pharmaceuticals, Inc.: Other, Research Funding. Off Label Use: Sorafenib as maintenance therapy for FLT3-ITD AML.

Sjögren Syndrome‐associated lymphomas: an update on pathogenesis and management

Primary Sjögren Syndrome (pSS) is an autoimmune disease associated with an increased risk of lymphoma. Lymphomas complicating pSS are mostly low-grade B cell non-Hodgkin lymphomas, predominantly of marginal zone histological type. Mucosal localization is predominant, notably mucosa-associated lymphoid tissue lymphomas. Lymphomas often develop in organs where pSS is active, such as salivary glands. Germinal centre (GC)-like structures, high TNFSF13B (BAFF) and Flt3-ligand (FLT3LG) levels and genetic impairment of TNFAIP3 are new predictors of lymphoma development. These new findings allow a better understanding of the pathogenic mechanisms leading to lymphoma. We propose the following scenario: auto-immune B cells with rheumatoid factor (RF) activity are continuously stimulated by immune complexes containing antibodies against more specific auto-antigens, such as SSA/Ro, SSB/La or others. Germline abnormality of TNFAIP3 leads to a decreased control of the NF-kB pathway and thus promotes survival of B cells and oncogenic mutations especially in GC structure. Moreover, B cells are stimulated by a positive loop of activation induced by BAFF secretion. Thus, lymphomagenesis associated with pSS exemplifies the development of antigen-driven B-cell lymphoma. The control of disease activity by a well-targeted immunosuppressor is the primary objective of the management of the patient in order to repress chronic B cell stimulation.

Flt3 ligand does not differentiate between Parkinsonian disorders.

Differential diagnosis of parkinsonian disorders is challenging because of overlapping symptoms, especially during early stages of disease. No validated biomarkers are available for early and accurate diagnosis of multiple system atrophy and other parkinsonian disorders. It has been reported that flt3 ligand levels in cerebrospinal fluid could clearly differentiate patients with Parkinson's disease from patients with multiple system atrophy, with 99% sensitivity and 95% specificity. We measured flt3 ligand levels in cerebrospinal fluid of subjects with Parkinson's disease (n = 37), multiple system atrophy (n = 30), and progressive supranuclear palsy (n = 19). In our cohort, no significant difference was found in flt3 ligand levels between Parkinson's disease, multiple system atrophy, and progressive supranuclear palsy. Our results suggest that cerebrospinal fluid flt3 ligand levels do not differentiate between parkinsonian disorders.

Low levels of SDF-1α constrain DC recovery and diminish homeostatic proliferation of naïve CD4+ T cells during graft-versus-host disease. (TRAN3P.892)

Abstract Graft-versus-host disease (GVHD) is the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (SCT) and its effect on T cell regeneration greatly increases the immunodeficiency normally associated with SCT. As a result, GVHD patients experience profound lymphopenia and lymphocyte reconstitution takes generally several months or years. The goal of this work was to assess the impact of GVHD on homeostatic proliferation (HP) of non-alloreactive naïve CD4+ T lymphocytes with the hypothesis that regeneration of the peripheral niche permissive to CD4 HP is constrained by GVHD. To study the impact of GVHD on the peripheral niche regulating CD4 HP, we used a Parent→F1 mouse model and observed a complete abrogation of CD4 HP in GVHD hosts. Loss of HP was related to depletion of all DC subsets as well as low levels of IL-7. While Flt3-ligand (FL) levels were normal, Stromal Derived Factor-1α (SDF-1α) was significantly diminished in GVHD mice. Administration of FL or SDF-1α significantly increased DC counts but CD4 HP was restored only when FL or SDF-1α was combined to IL-7. Importantly, while FL treatment aggravated GVHD, GVHD was improved by SDF-1α therapy. Our study shows that lack of immune reconstitution of CD4+ T cells is primarily due to failure of DC recovery owing to diminished SDF-1α production and diminished IL-7 and that the combination of SDF-1α and IL-7 could be used to enhance CD4 HP in GVHD hosts.

DOSE AND DOSE RATE EFFECTS OF IRRADIATION ON BLOOD COUNT AND CYTOKINE LEVEL IN BALB/c MICE

The biological effects of radiation are dependent on the dose rate and dose of radiation. In this study, effects of dose and dose rate using whole body radiation on plasma cytokines and blood count from male BALB/c mice were evaluated. We examined the blood and cytokine changes in mice exposed to a low (3.49 mGy h-1) and high (2.6 Gy min-1) dose rate of radiation at a total dose of 0.5 and 2 Gy, respectively. Blood from mice exposed to radiation were evaluated using cytokine assays and complete blood count. Peripheral lymphocytes and neutrophils decreased in a dose dependent manner following high dose rate radiation. The peripheral lymphocytes population remained unchanged following low dose rate radiation; however, the neutrophils population increased after radiation. The sera from these mice exhibited elevated levels of flt3 ligand and granulocyte-colony-stimulating factor (G-CSF), after high/low dose rate radiation. These results suggest that low-dose-rate radiation does not induce blood damage, which was unlike high-dose-rate radiation treatment; low-dose-rate radiation exposure activated the hematopoiesis through the increase of flt3 ligand and G-CSF.

Final Report Of Phase II Study Of Sorafenib and 5-Azacytidine In Patients With Relapsed Or Untreated Acute Myeloid Leukemia and FLT3-ITD mutation

BackgroundA potential mechanism of resistance to FLT3 kinase inhibitors is high levels of FLT3 ligand (FL) which is commonly seen after treatment with myelosuppressive chemotherapy. We hypothesized that combining sorafenib with a less myelosuppressive agent, such as 5-azacytidine (AZA), may lead to higher and more durable responses. Furthermore, differentiation of leukemic blasts has been reported with both agents. MethodsPatients were eligible if they had relapsed or refractory AML, were 18 years of age or older, and had adequate performance status (ECOG 2 2) and organ function. Patients older than 60 without prior therapy were also eligible if they were unsuitable to receive chemotherapy or refused it. Presence of FLT3-ITD was not a requirement but these patients were sought for participation in the study. Treatment regimen included AZA 75 mg/m2 daily for 7 days together with sorafenib 400 mg twice daily for 28 days; cycles were repeated in approximately 4 to 5-week intervals. Dose adjustments of both agents, and delay of AZA, based on toxicity were allowed. Plasma samples were collected on Day 1 and Day 10 of each cycle. ResultsOverall, 57 patients with AML with a median age of 65 years (range, 21-87) were enrolled. They included 27 (47%) patients with normal cytogenetics, 12 (21%) with chromosome 5/7 or complex cytogenetic abnormalities, 15 (26%) with other miscellaneous abnormalities; 3 (5%) had insufficient metaphases. Prior to the initiation of treatment, FLT3-ITD was detected in 53/57 (93%) patients with a median allelic ratio of 0.348 (range, 0.009 – 0.934). They had received a median of 2 prior treatments (range, 0 -7) including 13 previously untreated patients. Twenty (35%) patients had received ≥3 prior regimens and 12 had failed prior therapy with FLT3 kinase inhibitors (6 with AC220, 2 with PKC412, and 9 with sorafenib, either as monotherapy or with chemotherapy or plerixafor); 4 patients had failed 2 prior FLT3 inhibitors. The overall CR/CRi/PR rate among the 57 patients is 44%, including 16 (28%) with CRi and 8 (14%) with CR and 1 (2%) with PR. The response rate among the previously untreated patients was 62% and among relapsed patients 39%. Patients have received a median of 3 (range, 1 - 27) treatment cycles with the median number of cycles to response among the responders being 2 (range, 1 – 4) and the median time to achieving response, 2.1 months (range, 0.8 – 4.6 months). The median duration of CR/CRi Is 2.4 months (range, 0.8 – 24.8 months). Seven patients have proceeded to allogeneic stem cell transplant. The most common study drug-related adverse events were rash and fatigue with no deaths attributable to study medications. One patient developed grade 3 cardiomyopathy. With a median follow-up of 8.6 months (range, 6.1 – 26.9), 13 patients remain alive, 5 still in remission. The median overall survival of the 57 patients was 6.3 months, and 12.4 months in the 25 responding patients. Mean FL levels at cycle 2, day 0 and cycle 2, day 10 were 27 pg/mL and 54 pg/mL, respectively, which is significantly lower than those seen previously in studies of FLT3 kinase inhibitor plus chemotherapy. ConclusionsCombination of AZA and Sorafenib is effective for the treatment of patients with AML and FLT3-ITD. FL levels are significantly lower during the course of therapy with this combination compared to previously reported levels after chemotherapy. [Display omitted] Disclosures:Ravandi: Celgene: Consultancy, Research Funding.

Pharmacodynamic Analysis Of The Inhibitory Potency Of The Tyrosine Kinase Inhibitor Midostaurin In Combination With Intensive Chemotherapy Including Allogeneic Hematopoietic Stem Cell Transplantation Followed By Maintenance Therapy In FLT3-ITD Positive Acute Myeloid Leukemia In The Ongoing AMLSG 16-10 Trial

BackgroundActivating mutations in the receptor tyrosine kinase FLT3 occur in roughly 30% of acute myeloid leukemia (AML) patients (pts), implicating FLT3 as a potential target for kinase inhibitor therapy. The multi-targeted kinase inhibitor midostaurin (PKC412) shows potent activity against FLT3 as a single agent but also in combination with intensive chemotherapy. Besides its mere presence, the allelic ratio as well as ITD insertion site within the FLT3 gene had been reported as prognostic factors in FLT3-ITD positive AML. Furthermore, pharmacokinetic analyses revealed clinically important interactions between potent CYP3A4 inhibitors, such as azoles, and midostaurin. AimsTo evaluate the pharmacodynamic activity of midostaurin measured as inhibition of the degree of phosphorylated FLT3 (pFLT3) in correlation to co-medication and outcome data. MethodsThe study includes intensively treated adults (age 18-70 years) with newly diagnosed FLT3-ITD positive AML enrolled in the ongoing single-arm phase-II AMLSG 16-10 trial (NCT: NCT01477606). Pts with acute promyelocytic leukemia are not eligible. The presence of FLT3-ITD is analyzed by Genescan-based fragment-length analysis (allelic ratio >0.05 required to be FLT3-ITD positive). Induction therapy consists of daunorubicin (60 mg/m², d1-3) and cytarabine (200 mg/m², continuously, d1-7); midostaurin 50 mg twice daily is applied from day 8 onwards until 48h before start of the next treatment cycle. For consolidation therapy, pts proceed to allogeneic hematopoietic stem cell transplantation (HSCT) as first priority; if allogeneic HSCT is not possible pts receive three cycles of age-adapted high-dose cytarabine in combination with midostaurin from day 6 onwards. In all pts maintenance therapy for one year is intended. A total sample size of n=142 is planned to show an improvement in event-free survival from 25% after 2 years to 37.5%. Plasma inhibitory activity assay (PIA) for pFLT3 is performed as previously described (Levis MJ, et al. Blood 2006; 108:3477-83). For PIA, measured time points include day 15 of induction therapy, the end of each treatment cycle and every three months during maintenance therapy. ResultsTo date, 72 pts (median age, 54.5 years; range, 29-69 years) have been included and PIA was performed so far in 37 pts during induction therapy. Median pFLT3 inhibition after one week of midostaurin intake measured on day 15 of cycle 1 (C1D15) was 57.5% (range, 14.2-93.7%) with 2 of 31 pts showing inhibition >85%. At the end of the first induction cycle (C1end), median inhibition was 60.3% (range, 0-99.8%); here, 6 of 37 pts had an inhibition >85%. Co-medication with azoles was present in 7 of 23 pts at C1D15 and 13 of 28 pts at C1end. There was no significant difference in pFLT3 inhibition either on C1D15 (p=0.79) or at C1end (p=0.70) between pts on (median pFLT3 inhibition: 52.5%) or off (median pFLT3 inhibition 57.5%) azoles. Response data were available in 56 pts: complete remission (CR) was achieved in 78.5%; rates of early death and refractory disease (RD) were 9% and 12.5%, respectively. In first analyses, there was no difference in pFLT3 inhibition in pts achieving CR (n=30) as compared to those with RD (n=3; p=0.99). In contrast to our previously published data from three historical trials without a FLT3 inhibitor which showed that high allelic ratio was associated with low CR rates (Kayser S, et al. Blood 2009;114:2386-92), in the current trial CR rates remained high (81.5%) despite of a high allelic ratio above the median (>0.58). In addition, we did not see a negative prognostic impact of ITD insertion site within the tyrosine kinase domain of the FLT3 gene (p=0.99). Analyses are currently ongoing, measurement of FLT3 ligand levels and evaluation of pharmacokinetics of midostaurin are also intended. ConclusionsThe addition of 50 mg midostaurin twice daily to intensive induction therapy resulted in a moderate pFLT3 inhibition during induction therapy. Nonetheless, CR rates are promising, especially in pts with unfavorable FLT3-ITD characteristics. Concomitant azoles do not appear to significantly influence pFLT3 inhibitory activity of midostaurin. Disclosures:Levis:Ambit Biosciences: Consultancy. Schlenk:Ambit: Honoraria; Chugai: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Amgen: Research Funding.

213Bi Radioimmunotherapy with an Anti-mCD138 Monoclonal Antibody in a Murine Model of Multiple Myeloma

New multiple myeloma (MM) treatments--such as high-dose melphalan therapy plus autologous stem cell transplantation or regimens incorporating bortezomide, thalidomide, and lenalidomide--substantially increase the rate of complete response that is associated with longer patient survival. Thus, maintaining the complete response status by improving the minimal residual disease after induction therapy is a key goal for MM management. Here, we address the question of radioimmunotherapy efficacy in MM minimal residual disease treatment in mice with a low tumor burden. α-emitters are particularly well adapted to this approach because the short range of α-particles enables localized irradiation of tumor cells within the bone marrow and a cytotoxic effect on isolated cells due to the high LET (linear energy transfer) of α-particles. The CD138 antigen was used as a target because of its strong expression on myeloma cells in 100% of patients. Intravenous injection of 10(6) 5T33 mouse myeloma cells into the Syngeneic mouse strain C57BL/KaLwRij resulted in a rapid invasion of the marrow and limb paralysis, as illustrated by bioluminescence imaging with luciferase-transfected 5T33 cells. Radioimmunotherapy was performed 10 d after 5T33 cell engraftment with an intravenous injection of an antimouse CD138 antibody radiolabeled with (213)Bi at activities of 1.85, 3.7, 7.4, and 11.1 MBq. A blood cell count was performed weekly to monitor hematologic toxicity. The levels of blood Flt3 ligand were also measured to evaluate the radioimmunotherapy-related myelotoxicity. Disease progression was monitored by titrating the monoclonal IgG2b antibody produced by 5T33 cells. The groups treated with 3.7 and 7.4 MBq exhibited a median survival greater than 300 and 227 d, respectively, compared with 45.5 d in the control untreated group. The highest activity (11.1 MBq) showed short-term toxicity whereas the lowest activity (1.85 MBq) gave results similar to those of the controls. With activities of 3.7 and 7.4 MBq, mice exhibited a transient hematologic toxicity whereas only temporary and moderate myelotoxicity was observed with 7.4 MBq. This study demonstrates promising therapeutic efficacy of (213)Bi-labeled anti-mCD138 for the treatment of residual disease in the case of MM, with only moderate and transient toxicity.

THU0090 Survivin but not FMS-like Tyrosine Kinase Ligand (FLT3L) is Up-Regulated Before Onset of Rheumatoid Arthritis

BackgroundPresence of antibodies against citrullinated peptides (anti-CCP2) and increased levels of cytokines and chemokines precedes the development of rheumatoid arthritis (RA) by several years [1, 2]. Recently, the proteins survivin...

Variations in FLT3 ligand levels during the course of AML treatment.

7026 Background: FLT3 ligand (FL) is a hematopoietic growth factor expressed in many tissues. AML patients who are administered myeloablative therapy exhibit a marked and transient rise in plasma FL concentrations. Furthermore, the presence of high concentrations (1000 pg/mL) of FL impedes the efficacy of FLT3 tyrosine kinase inhibitors in vitro. However, the behavior of FL concentrations throughout the course of AML treatment remains unknown. This pilot study was undertaken to track the relationship between AML therapy and FL levels over time. Methods: Ten AML patients were enrolled in an IRB-approved procurement protocol. Blood samples were collected at weekly intervals for one year, and plasma was isolated by centrifugation. Plasma FL and stem cell factor (SCF) concentrations were measured by ELISA. Results: We observed four distinct patterns in FL fluctuations. First, in all cases where induction or consolidation chemotherapy resulted in an aplastic bone marrow (nine patients), FL concentrations rose markedly and consistently to levels &gt;1000 pg/mL following the administration of chemotherapy. Second, in three of four patients whose leukemia was refractory to induction chemotherapy, FL concentrations remained below 500 pg/mL during induction. Third, in two patients receiving the FLT3 TKI sorafenib, FL concentrations did not rise above 500 pg/mL while on this medication. Fourth, in one patient receiving the hypomethylating agent 5-azacitidine, FL concentrations remained below 100 pg/mL throughout the course of therapy. SCF concentrations did not vary throughout the course of chemotherapy. Conclusions: An “FL surge” was seen when cytotoxic chemotherapy resulted in aplasia. This FL surge was not seen with sorafenib or 5-azacitidine. In addition, the FL surge was attenuated in three patients whose leukemia was refractory to chemotherapy. These observations give rise to two new hypotheses regarding FL: 1) It is possible to maintain lower FL levels with targeted agents than with chemotherapy; and 2) Residual leukemia appears to inhibit the FL surge, providing indirect evidence of cross-talk between leukemia and the stromal microenvironment. This inhibition may be the explanation for why AML patients develop pancytopenia early in relapse.

Correlation of Plasma FL Expression with Bone Marrow Irradiation Dose

PurposeAblative bone marrow irradiation is an integral part of hematopoietic stem cell transplantation. These treatment regimens are based on classically held models of radiation dose and the bone marrow response. Flt-3 ligand (FL) has been suggested as a marker of hematopoiesis and bone marrow status but the kinetics of its response to bone marrow irradiation has yet to be fully characterized. In the current study, we examine plasma FL response to total body and partial body irradiation in mice and its relationship with irradiation dose, time of collection and pattern of bone marrow exposure.Materials/MethodsC57BL6 mice received a single whole body or partial body irradiation dose of 1–8 Gy. Plasma was collected by mandibular or cardiac puncture at 24, 48 and 72 hr post-irradiation as well as 1–3 weeks post-irradiation. FL levels were determined via ELISA assay and used to generate two models: a linear regression model and a gated values model correlating plasma FL levels with radiation dose.ResultsAt all doses between 1–8 Gy, plasma FL levels were greater than control and the level of FL increased proportionally to the total body irradiation dose. Differences in FL levels were statistically significant at each dose and at all time points. Partial body irradiation of the trunk areas, encompassing the bulk of the hematopoietically active bone marrow, resulted in significantly increased FL levels over control but irradiation of only the head or extremities did not. FL levels were used to generate a dose prediction model for total body irradiation. In a blinded study, the model differentiated mice into dose received cohorts of 1, 4 or 8 Gy based on plasma FL levels at 24 or 72 hrs post-irradiation.ConclusionOur findings indicate that plasma FL levels might be used as a marker of hematopoietically active bone marrow and radiation exposure in mice.

481: Maternal-fetal cellular trafficking and fetal inflammation in patients with preterm premature rupture of membranes

inflammation in patients with preterm premature rupture of membranes Dana Henry, Yvonne Cheng, Payam Saadai, Sheila Keating, Tzong-Hae Lee, Philip Norris, Michael Busch, Roberto Romero, Tippi MacKenzie UCSF, Obstetrics and Gynecology, San Francisco, CA, UCSF, Surgery, San Francisco, CA, Blood Systems Research Institute, Blood Bank, San Francisco, CA, Hutzel Women’s Hostpial, Obstetrics and Gynecology, Detroit, MI OBJECTIVE: Maternal-fetal cellular trafficking (MFCT) is the bidirectional passage of cells between the mother and her fetus. We previously reported alterations in trafficking after fetal surgery but the extent of MFCT in preterm premature rupture of membranes (PPROM) has not been characterized. We studied MFCT and changes in potential biomarkers in maternal and umbilical cord blood in PPROM. STUDY DESIGN: We prospectively obtained matched maternal and umbilical cord blood samples from 23 healthy term control patients and 13 patients with PPROM. The extent of MFCT was determined using PCR for non-shared HLA-DR alleles. “High traffickers” were defined as those above the 75th percentile of control patients. Serum cytokine and chemokine levels were analyzed using a human 39plex immunoassay system (Millipore). RESULTS: The gestational age at delivery was lower in patients with PPROM than in the control group (33.1 vs. 39.5 wks, p 0.01 by t-test). High trafficking of maternal cells into cord blood was more common in patients with PPROM than in those in the control group (60% vs. 23%, P 0.007 by Chi-square test), while trafficking of fetal cells in maternal blood was unchanged. Fetal levels of Flt-3 ligand, IL-1 , IL-1R , IL-10, monocyte chemotactic protein-1, and sIL-2R were significantly higher in PPROM, while levels of macrophage-derived chemokine were significantly lower (p 0.05 by Kruskal-Wallis). Maternal levels of IL-6 and IL-10 were increased and levels of eotaxin were decreased in PPROM compared to controls. Patients with PPROM and high trafficking had significantly higher concentrations of the chemokine MIP-1 (CCL4) in cord blood than those with low trafficking, suggesting a mechanism for recruitment of maternal cells. CONCLUSION: This is the first demonstration of high MFCT in PPROM, with coordinate changes in inflammatory and chemotactic cytokines in fetal blood. Our observations suggest a fetal origin of labor in PPROM and indicate potential new targets for therapeutic intervention. 482 Placental histologic findings of chorion-decidual hemorrhage and inflammation in spontaneous preterm birth influence outcomes in the subsequent pregnancy David Hackney, Radhika Tirumala, Linda Salamone, Richard Miller, Philip Katzman University of Rochester School of Medicine, Obstetrics and Gynecology, Rochester, NY, University of Rochester School of Medicine, Pathology, Rochester, NY, Rochester General Hosptial, Obstetrics and Gynecology, Rochester, NY OBJECTIVE: Spontaneous preterm birth (SPTB) is a common endpoint of different underlying etiologies, including inflammation and chorion-decidual bleeding. The presumed etiology from placental histology may influence outcomes in subsequent pregnancies. STUDY DESIGN: Included subjects had: 1) a SPTB after 2004 with available placental pathology and 2) a subsequent delivery at 20 weeks EGA at our institution. Exclusion criteria were multifetal gestation, thrombophilia, anticoagulation or substance abuse. For included subjects archived placenta and membrane paraffin blocks from the index SPTB were cut, stained with Prussian Blue and evaluated by a perinatal pathologist for the presence of hemosiderin. The association between histologic findings and subsequent pregnancy outcomes were evaluated through logistic and linear regression. RESULTS: A total of 131 subjects were included, of whom 39.7% had a recurrent SPTB. The index SPTB demonstrated chorioamnionitis in 42.7%, vasculitis or funisitis in 24.4% and chorion-decidual bleeding Unadjusted and propensity score (PS)-adjusted risk ratios (95% confidence interval) of respiratory distress syndrome (RDS) and neonatal sepsis by antenatal corticosteroid (ACS) exposure and gestational age at delivery Levels of cytokines and chemokines in umbilical cord blood of normal term control patients and those with PPROM Poster Session III Doppler Assess, Fetus, Prematurity, U/S, Med-Surg-Diseases www.AJOG.org

Combination of Sorafenib and 5-Azacytidine Has Significant Activity in Patients with Relapsed/Refractory or Untreated Acute Myeloid Leukemia and FLT3-ITD mutation

Abstract 1519▪▪This icon denotes a clinically relevant abstract Background:The outcome of patients (pts) with acute myeloid leukemia (AML) and FLT3-ITD mutation is poor, particularly in the relapse setting. Sorafenib is a potent inhibitor of FLT3 kinase with reported clinical activity as a single agent (Metzelder S, Blood, 2009), and in combination with chemotherapy (Ravandi F, JCO, 2010). A potential mechanism of resistance to FLT3 kinase inhibitors is high levels of FLT3 ligand (FL) as seen after myelosuppressive chemotherapy. We hypothesized that combining sorafenib with a less myelosuppressive agent, such as 5-azacytidine (AZA), may lead to higher and more durable responses than cytotoxic chemotherapy. Furthermore, both drugs have demonstrated a potential for inducing differentiation in AML cells, thereby providing further rationale for the combination. Methods:Pts were eligible if they had relapsed or refractory AML, were 18 years of age or older, and had adequate performance status (ECOG ≤ 2) and organ function. Older pts without prior therapy were also eligible, if they were deemed unsuitable to receive chemotherapy. Presence of FLT3-ITD was not a requirement but these pts were targeted for enrollment. Treatment regimen included AZA 75 mg/m2 daily for 7 days together with sorafenib 400 mg twice daily for 28 days; cycles were repeated in approximately 4 to 5-week intervals. Overall responses were assessed after the completion of at least one cycle of therapy and at the time of the best peripheral blood and bone marrow response. Plasma samples were collected on approximately Day 1 and Day 10 of each cycle. To assess the degree of FLT3 inhibition, the plasma inhibitory activity (PIA) assay was performed using the Molm-14 cell line (Levis M, Blood, 2006). Plasma FL concentrations were measured using an ELISA kit (R&D Systems). Results:43 pts with AML with a median age of 64 years (range, 24–87) were enrolled. They included 19 (44%) pts with diploid cytogenetics, 11 (26%) with chromosome 5/7 or complex cytogenetic abnormalities, and 13 (30%) with miscellaneous abnormalities. Prior to the initiation of treatment, FLT3-ITD was detected in 40/43 (93%) pts with a median allelic ratio of 0.28 (range, 0 – 0.93). They had received a median of 2 prior treatments (range, 0–7). 16 (37%) pts had received ≥3 prior regimens and 9 had failed therapy with FLT3 kinase inhibitors (5 with AC220, 1 with PKC412, and 6 with sorafenib, either as monotherapy or with chemotherapy or plerixafor); 3 had failed 2 prior FLT3 inhibitors. 6 pts were inevaluable as they discontinued therapy before response assessment at one month and 3 are too early for response assessment. The overall CR/CRi/PR rate among the 34 evaluable pts is 44%, including 10 (29%) with CRi and 4 (12%) with CR and 1 (3%) with PR (in this pt, bone marrow blast declined from 51% to 6% with normalization of blood counts). Overall, pts have received a median of 3 (range, 1–9) treatment cycles with the median number of cycles to response among the responders being 2 (range, 1 – 4) and the median time to achieving response, 2.1 months (range, 0.9 – 4.6 months). The median duration of CR/CRi Is 2.3 months (range, 1 – 12.2+ months). Six pts have proceeded to allogeneic stem cell transplant. The most common study drug-related adverse events were rash and fatigue with no deaths attributable to study medications. One pt developed grade 3 cardiomyopathy suspected to be related to the study regimen. Of the 34 pts included in the clinical analysis, there were 22 pts from whom plasma samples spanning at least one cycle of therapy were available. Among them, 64% achieved FLT3 inhibition to a targeted level of less than 15% of baseline during their first cycle of therapy. Median survival in pts who achieved this degree of inhibition was 238 days, while median survival in pts who did not reach this level was 154 days (p=0.13). Mean FL levels at cycle 1, day 1 and cycle 1, day 10 were 9 pg/mL and 17 pg/mL, respectively. Mean FL levels at cycle 2, day 0 and cycle 2, day 10 were 27 pg/mL and 54 pg/mL, respectively. Conclusions:Combination of AZA and Sorafenib is effective for the treatment of older pts and pts with relapsed and refractory AML and FLT3-ITD mutation. While not statistically significant, there was a trend toward improved survival in pts with adequate FLT3 inhibition during cycle 1. FL levels did not rise to the levels seen in pts receiving cytotoxic chemotherapy. Disclosures:Ravandi:Bayer/Onyx: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Off Label Use: Off-label use of sorafenib and 5-azacytidine in patients with acute myeloid leukemia. Levis:Astellas Pharma: Consultancy; Plexxikon: Consultancy; Symphogen: Consultancy. Garcia-Manero:Celgene: Research Funding. Andreeff:Hoffmann-La Roche: Research Funding; Karyopharm Therapeutics: Unrestricted gift, Unrestricted gift Other. Cortes:Celgene: Research Funding.

Flt3 inhibitor AC220 is a potent therapy in a mouse model of myeloproliferative disease driven by enhanced wild-type Flt3 signaling

AbstractHigh levels of expression of wild-type Flt3 characterize many hematopoietic proliferative diseases and neoplasms, providing a potential therapeutic target. Using the c-Cbl RING finger mutant mouse as a model of a myeloproliferative disease (MPD) driven by wild-type Flt3, in the present study, we show that treatment with the Flt3 kinase inhibitor AC220 blocks MPD development by targeting Flt3+ multipotent progenitors (MPPs). We found that daily administration of AC220 caused a marked reduction in Flt3 expression, induction of quiescence, and a significant loss of MPPs within 4 days. Unexpectedly, a robust Flt3 ligand–associated proliferative recovery response soon followed, preventing further loss of MPPs. However, continued AC220 treatment limited MPP recovery and maintained reduced, steady-state levels of cycling MPPs that express low levels of Flt3. Therefore, a finely tuned balance between the opposing forces of AC220 and Flt3 ligand production was established; whereas the Flt3 ligand blunted the inhibitory effects of AC220, the disease was held in remission for as long as therapy was continued. The net effect is a potent therapy indicating that patients with c-Cbl mutations, or those with similarly enhanced Flt3 signaling, may respond well to AC220 even after the induction of high levels of Flt3 ligand.

Response rates in patients with relapsed/refractory acute myeloid leukemia with FLT3-ITD mutation using 5-azacitadine plus sorafenib.

24 Background: Outcome of patients with relapsed acute myeloid leukemia (AML) with FLT3-ITD mutation is poor. Elevated FLT3 ligand levels (FL) secondary to cytotoxic chemotherapy is a putative mechanism of resistance to the FLT3 kinase inhibitors. We hypothesized that FL levels will be lower when combining sorafenib with less intensive therapy such as 5-Azacytidine (5-Aza). This study was conducted to evaluate the efficacy and tolerability of the combination of sorafenib and 5-Aza in patients with refractory or relapsed AML with FLT3-ITD mutation. Methods: Patients were eligible if they had relapsed or refractory AML and were 18 years of age or older. They received 5-Aza 75 mg/m2 IV daily x 7 days and sorafenib 400 mg PO BID continuously; cycles were repeated in approximately one month intervals. Results: 38 patients with AML with a median age of 60 years (range, 24-87) were enrolled. 4 were inevaluable as they never received therapy or discontinued it before response assessment. FLT-3-ITD was detected in 30 (88%) patients. They had received a median of 2 prior treatments (range, 0-6); 13 (38%) had received ≥ 3 prior regimens and 8 had failed prior FLT3 kinase inhibitor. Response rate was 41%, including 6 (17%) with CRi, 4 (12) with CRp, 3 (9%) with CR, and 1(3%) PR. The median time to achieving CR/CRi was 2 cycles (Range, 1-6) and the median duration of CR/CRi was 3 months (Range, 1–12). Conclusions: Combination of 5-Aza and sorafenib is effective for patients with relapsed AML with FLT-3-ITD mutation.

Assessment of Total- and Partial-Body Irradiation in a Baboon Model

This biodosimetry study used irradiated baboons to investigate the efficacy of a kinetic multiparameter (clinical, physical, and biological) approach for discriminating partial-body irradiation (PBI) and total-body irradiation (TBI). Animals were unilaterally (front) exposed to 60Co gamma rays (8 to 32 cGy min) using either TBI or vertical left hemi-body irradiation (HBI), as follows: 2.5 Gy TBI (n = 2), 5 Gy TBI (n = 2), 5 Gy HBI (n = 2), and 10 Gy HBI (n = 2). Midline tissue doses were measured at the anterior iliac crest level with an ionization chamber, and body dosimetry was performed using thermoluminescent dosimeters. Blood samples were collected before exposure and from 1 h until 200 d after irradiation. Clinical status, complete blood cell count, biochemical parameters, and cytogenetic analysis were evaluated. The partial least square discriminant analysis chosen for statistical analysis showed that the four groups of irradiated baboons were clearly separated. However, the dicentric chromosome assay may not distinguish HBI from TBI in confounding situations where equivalent whole-body doses are similar and the time of exposure is sufficient for peripheral blood lymphocyte homogenization. Interestingly, as bone marrow shielding in HBI animals prevented aplasia from happening, hematologic parameters such as the platelet count and Flt-3 ligand level helped to distinguish HBI and TBI. Moreover, the ratio of neutrophil to lymphocyte counts, creatine kinase, and citrulline levels may be discriminating biomarkers of dose or injury. Both early and delayed clinical signs and bioindicators appear to be useful for assessment of heterogeneous irradiation.

Metastasin S100A4 is increased in proportion to radiographic damage in patients with RA

To assess the potential of metastasin S100A4 as a biological marker in patients with RA. A total of 87 unselected patients with established RA (disease duration 2-44 years) and treated with MTX and infliximab at a single rheumatology centre were included in a cross-sectional study. Radiographs of hands and feet were taken prior to infliximab treatment and at inclusion (time interval 48 ± 27 months) and scored for the radiographic damage. S100A4 levels were analysed in relation to radiographic damage, clinical disease activity (DAS-28), inflammation (IL-6, CRP, ESR), bone and cartilage markers [MMP-3, COMP, C-telopeptide of type I collagen (CTX-I)] and proto-oncogenes [survivin, insulin-like growth factor 1 (IGF-1), Flt3 ligand]. High levels of S100A4 were associated with severe radiographic damage (OR = 3.40, P = 0.025), non-response to infliximab (OR = 4.63, P = 0.003), presence of antibodies to infliximab (OR = 6.24, P = 0.003) and high levels of Flt3 ligand (OR = 2.73, P = 0.04). Regression analysis showed that high S100A4 was predictive for radiographic progression during infliximab treatment [positive predictive value (PPV) 0.68, P = 0.05]. Low levels of S100A4 were associated with response to infliximab (OR = 2.67, P = 0.049), clinical remission (OR = 4.01, P = 0.0047) and negative RF (OR = 9.22, P = 0.0047). S100A4 correlated with survivin (r = 0.71, P > 0.0001). S100A4 levels are increased in proportion to radiographic damage and its further progression in RA patients. High S100A4 levels were associated with a poor clinical response to infliximab and high rate of anti-infliximab antibodies. The finding of a correlation between S100A4 and survivin and Flt3 ligand suggests that these proteins may represent a new cluster of biomarkers predicting radiographic progression and poor treatment response in RA patients.