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Background: Overexpression of fms-like tyrosine kinase 3 (FLT3) protein in leukemia is highly related to poor prognosis and reduced survival rate in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Simple but efficient quantification of FLT3 protein levels on the leukemic cell surface using flow cytometry had been developed for rapid determination of FLT3 on intact cell surface., Methods: Quantitation protocol for FLT3 biomarker in clinical samples was developed and validated. Cell model selection for calibration curve construction was identified and evaluated. Selected antibody concentrations, cell density, and incubation time were evaluated for most appropriate conditions. Comparison of the developed FLT3 determination protocol with the conventional Western blot analysis was performed., Results: EoL-1 cell line was selected for using as positive control cells. Calibration curve (20%-120% of FLT3 positive cells) and quality control (QC) levels were constructed and evaluated. The results demonstrated good linearity (r 2  > 0.99). The intra- and inter-day precision and accuracy, expressed as the coefficient of variation (%CV) and % recovery, were <20% and fell in 80%-120% in all cases. When compared with Western blotting results, FLT3 protein expression levels in leukemia patient's bone marrow samples were demonstrated in the same trend., Conclusions: The effective, reliable, rapid, and economical analytical technique using the developed flow cytometric method was demonstrated for FLT3 protein determination on leukemic cell surface. This method provided a practical analysis of FLT-3 biomarker levels which is valuable for physician decision in acute leukemia treatment., (© 2019 The Authors. Journal of Clinical Laboratory Analysis Published by Wiley Periodicals, Inc.)

Allogeneic hematopoietic stem cell transplantation is an established consolidation therapy for patients with acute myeloid leukemia. However, relapse after transplantation remains a major clinical problem resulting in poor prognosis. Thus, detection of measurable ("minimal") residual disease to identify patients at high risk of relapse is essential. A feasible method to determine measurable residual disease may be digital droplet PCR (ddPCR) that allows absolute quantification with high sensitivity and specificity without the necessity of standard curves. Using ddPCR, we analyzed pre-transplant peripheral blood and bone marrow of 51 NPM1-mutated acute myeloid leukemia patients transplanted in complete remission or complete remission with incomplete recovery. Mutated NPM1 measurable residual disease-positive patients had higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.014). Restricting the analyses to patients receiving non-myeloablative conditioning, mutated NPM1 measurable residual disease positivity is associated with higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.006). Positive mutated NPM1 measurable residual disease status determined by ddPCR before allogeneic stem cell transplantation is associated with worse prognosis independent of other known prognostic markers-also for those receiving non-myeloablative conditioning. In the future, mutated NPM1 measurable residual disease status determined by ddPCR might guide treatment and improve patients' outcomes.

Background: The significance of FMS-like tyrosine kinase 3 (FLT3)-ITD mutation in acute myeloid leukemia (AML) prognosis has been well established. The aims of this study were to investigate the prognostic impact of the FLT3 protein (CD135) expression and its association with FLT3-ITD mutation, and to identify its role in minimal residual disease., Patients and Methods: CD135 was measured by flow cytometry on leukemic blasts of 257 adults with de novo AML. High expression of CD135 ≥ 20% was correlated with clinical, laboratory, and other prognostic factors that influenced treatment outcome. FLT3-ITD mutation was tested by PCR., Results: The frequency of CD135 expression was 138 (53.7%) of 257. FLT3-ITD was detected in (21.4%). Positive CD135 expression was associated with high total leukocyte count (P = .006), platelet count (P = .003), monocytic leukemia (P < .001), and CD34 (P = .008) and CD117 (P = .006) expression. CD135 expression ≥ 25% was a predictor of FLT3-ITD mutation (P = .03). CD135 overexpression was a negative predictor of complete remission and of postinduction minimal residual disease at days 14 and 28 (P < .001). CD135 had an adverse impact on overall and disease-free survival (68.5% vs. 15%, P = .002). Multivariate analysis indicated CD135 was the sole independent prognostic factor for overall survival (hazard ratio = 2.49; 95% confidence interval, 1.855-3.345; P < .001)., Conclusion: CD135 is emerging as a prognostic factor, a new marker for minimal residual disease, and a potential novel therapeutic target of AML., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+ cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML., Competing Interests: The authors have no conflicts to declare.

Many promising new cancer drugs proceed through preclinical testing and early-phase trials only to fail in late-stage clinical testing. Thus, improved models that better predict survival outcomes and enable the development of biomarkers are needed to identify patients most likely to respond to and benefit from therapy. Here, we describe a comprehensive approach in which we incorporated biobanking, xenografting, and multiplexed phospho-flow (PF) cytometric profiling to study drug response and identify predictive biomarkers in acute myeloid leukemia (AML) patients. To test the efficacy of our approach, we evaluated the investigational JAK2 inhibitor fedratinib (FED) in 64 patient samples. FED robustly reduced leukemia in mouse xenograft models in 59% of cases and was also effective in limiting the protumorigenic activity of leukemia stem cells as shown by serial transplantation assays. In parallel, PF profiling identified FED-mediated reduction in phospho-STAT5 (pSTAT5) levels as a predictive biomarker of in vivo drug response with high specificity (92%) and strong positive predictive value (93%). Unexpectedly, another JAK inhibitor, ruxolitinib (RUX), was ineffective in 8 of 10 FED-responsive samples. Notably, this outcome could be predicted by the status of pSTAT5 signaling, which was unaffected by RUX treatment. Consistent with this observed discrepancy, PF analysis revealed that FED exerted its effects through multiple JAK2-independent mechanisms. Collectively, this work establishes an integrated approach for testing novel anticancer agents that captures the inherent variability of response caused by disease heterogeneity and in parallel, facilitates the identification of predictive biomarkers that can help stratify patients into appropriate clinical trials., (©2016 American Association for Cancer Research.)

Activating mutations of FMS-like tyrosine kinase 3 (FLT3), notably internal tandem duplications (ITDs), are associated with a grave prognosis in acute myeloid leukemia (AML). Transforming FLT3ITD signal transduction causes formation of reactive oxygen species (ROS) and inactivation of the protein-tyrosine phosphatase (PTP) DEP-1/PTPRJ, a negative regulator of FLT3 signaling. Here we addressed the underlying mechanisms and biological consequences. NADPH oxidase 4 (NOX4) messenger RNA and protein expression was found to be elevated in FLT3ITD-positive cells and to depend on FLT3ITD signaling and STAT5-mediated activation of the NOX4 promoter. NOX4 knockdown reduced ROS levels, restored DEP-1 PTP activity and attenuated FLT3ITD-driven transformation. Moreover, Nox4 knockout (Nox4(-/-)) murine hematopoietic progenitor cells were refractory to FLT3ITD-mediated transformation in vitro. Development of a myeloproliferative-like disease (MPD) caused by FLT3ITD-transformed 32D cells in C3H/HeJ mice, and of a leukemia-like disease in mice transplanted with MLL-AF9/ FLT3ITD-transformed murine hematopoietic stem cells were strongly attenuated by NOX4 downregulation. NOX4-targeting compounds were found to counteract proliferation of FLT3ITD-positive AML blasts and MPD development in mice. These findings reveal a previously unrecognized mechanism of oncoprotein-driven PTP oxidation, and suggest that interference with FLT3ITD-STAT5-NOX4-mediated overproduction of ROS and PTP inactivation may have therapeutic potential in a subset of AML.

Background: Internal tandem duplication of FMS-like tyrosine kinase (FLT3-ITD) is well known to be involved in acute myeloid leukemia (AML) progression, but FLT3-ITD-negative AML cases account for 70% to 80% of AML, and the mechanisms underlying their pathology remain unclear. This study identifies protein tyrosine phophatase PRL-3 as a key mediator of FLT3-ITD-negative AML., Methods: A total of 112 FLT3-ITD-negative AML patients were sampled between 2010 and 2013, and the occurrence of PRL-3 hyperexpression in FLT3-ITD-negative AML was evaluated by multivariate probit regression analysis. Overexpression or depletion of endogenous PRL-3 expression with the specific small interfering RNAs was performed to investigate the role of PRL-3 in AML progression. Xenograft models were also used to confirm the oncogenic role of PRL-3., Results: Compared to healthy donors, PRL-3 is upregulated more than 3-fold in 40.2% of FLT3-ITD-negative AML patients. PRL-3 expression level is adversely correlated to the overall survival of the AML patients, and the AML relapses accompany with re-upregulation of PRL-3. Mechanistically, aberrant PRL-3 expression promoted cell cycle progression and enhanced the antiapoptotic machinery of AML cells to drug cytotoxicity through downregulation of p21 and upregulation of Cyclin D1 and CDK2 and activation of STAT5 and AKT. Depletion of endogenous PRL-3 sensitizes AML cells to therapeutic drugs, concomitant with apoptosis by upregulation of cleaved PARP (poly ADP ribose polymerase) and apoptosis-related caspases. Xenograft assays further confirmed PRL-3's oncogenic role in leukemogenesis., Conclusions: Our results demonstrated that PRL-3 is a novel independent crucial player in both FLT3-ITD-positive and FLT3-ITD-negative AML and could be a potential therapeutic target., (© 2014 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society.)

There is lack of data with regard to FLT3 expression in FLT3-ITD positive pediatric AML patients. Further, FLT3-ITD has not been systematically analyzed for outcome from Indian subcontinent. Amongst 64 consecutive pediatric AML patients, FLT3-ITD was present in 12 (19%) patients. All patients with FLT3-ITD achieved CR; those with FLT3-ITD mutation had inferior DFS (P = .029). FLT3 expression by flow-cytometry was observed in all FLT3-ITD positive patients, whereas 40/52 (77%) FLT3-ITD negative patients expressed FLT3 (P = .06). FLT3 expression in 12 FLT3-ITD positive patients was unable to show an association between FLT3 expression and outcome. In FLT3-ITD negative patients, higher surface expression of FLT3 significantly predicted poor EFS (P = .001) and OS (P = .007).

The suppressor of cytokine signaling 2 (SOCS2) is a member of the SOCS family of E3 ubiquitin ligases. SOCS2 is known to regulate signal transduction by cytokine receptors and receptor tyrosine kinases. The receptor tyrosine kinase FLT3 is of importance for proliferation, survival and differentiation of hematopoietic cells and is frequently mutated in acute myeloid leukemia. We observed that SOCS2 associates with activated FLT3 through phosphotyrosine residues 589 and 919, and co-localizes with FLT3 in the cell membrane. SOCS2 increases FLT3 ubiquitination and accelerates receptor degradation in proteasomes. SOCS2 negatively regulates FLT3 signaling by blocking activation of Erk 1/2 and STAT5. Furthermore, SOCS2 expression leads to a decrease in FLT3-ITD-mediated cell proliferation and colony formation. Thus, we suggest that SOCS2 associates with activated FLT3 and negatively regulates the FLT3 signaling pathways., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The classification of acute myeloid leukemia (AML) has evolved to the most recent World Health Organization (WHO) schema, which integrates genetic, morphologic, and prognostic data into a single system. However, this system was devised using adult data and how this system applies to a pediatric cohort is unknown. Performing a retrospective chart review, we examined our single-center experience with AML in 115 children and classified their leukemia using the WHO 2008 schema. We examined patient samples for mutations of FLT3, NPM1, and CEBPA. Overall survival was calculated within categories. In our pediatric population, most cases of AML had recurrent genetic abnormalities of favorable prognosis. More than 10% of patients in our series were categorized as AML, with myelodysplasia-related changes, an entity not well-described in pediatric patients. In addition, a large proportion of patients were categorized with secondary, therapy-related AML. To our knowledge, this is the first application of the WHO 2008 classification to a pediatric cohort. In comparison to adult studies, AML in the pediatric population shows a distinct distribution within the WHO 2008 classification.

A 6-year-old boy was diagnosed with FLT3-ITD+acute monoblastic leukemia (AMoL). He showed resistance to 2 cycles of induction chemotherapy with etoposide, cytarabine, and mitoxantrone or idarubicin performed according to the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 protocol. His condition was also refractory to salvage FLAI-GO (fludarabine, cytarabine, idarubicin, and gemtuzumab ozogamicin) chemotherapy. Sequential administration of sorafenib at doses of up to 300 mg/day resulted in the first remission. He underwent bone marrow transplantation from his HLA 2-locus mismatched father. Recurrence was observed on post-transplantation day 71. A sustained partial response was observed after alternate-day readministration of sorafenib 150mg/day. In spite of a donor lymphocyte infusion, his blast cell count increased on day 245. Chemotherapy with an increased dose of sorafenib reduced the blast cell count. Although a second HLA-mismatched allogeneic peripheral blood stem cell transfusion was performed, the patient died of regimen-related toxicity. Herein, we report a pediatric case of primary refractory FLT3-ITD+ AMoL. Further prospective studies are necessary to validate the efficacy of sorafenib treatment.

The impact of a FLT3-internal tandem duplication (FLT3ITD) on prognosis of patients with acute myeloid leukemia (AML) is dependent on the ratio of mutated to wild-type allele. In 648 normal karyotype (NK) AML patients, we found a significant independent effect of the quantitative FLT3ITD mRNA level--measured as (FLT3ITD/wtFLT3)/(FLT3ITD/wtFLT3+1)--on outcome. Moreover, this effect was clearly seen in 329 patients with a mutated NPM1 gene (NPM1+), but not in 319 patients without a NPM1 mutation (wtNPM1). In a multivariate Cox regression model, the quantitative FLT3ITD mRNA level showed an independent prognostic impact on overall survival (OS) and relapse-free survival (RFS) only in the NPM1+ subgroup (OS: hazard ratio, 5.9; [95% confidence interval [CI]: 3.1-11.2]; RFS: hazard ratio, 7.5 [95% CI: 3.4-16.5]). The FLT3ITD mRNA level contributes to relapse risk stratification and might help to guide postremission therapy in NPM1-mutated AML.

We reviewed the frequency and prognostic significance of FLT3 (fms-like tyrosine kinase receptor-3) and NPM (nucleophosmin) gene mutations and WT1 (Wilms' tumor) and BAALC (brain and acute leukemia, cytoplasmic) gene expression in 100 consecutive patients with intermediate and poor cytogenetic risk de novo acute myeloid leukemia (AML) receiving conventional anthracycline-AraC based therapy. We observed a strict relationship between unfavorable karyotype and BAALC >1000 (p = 0.0001). Multivariate analysis of 81 patients with intermediate karyotype revealed that younger age (p = 0.00009), NPM gene mutation (p = 0.002), and WT1 >75th percentile (>2365) (p = 0.003) were independent, positive factors for complete remission (CR). WT1 expression above 2365 was correlated also to longer event-free survival (EFS) and overall survival (OS) in the same subset of patients (p = 0.003 and p = 0.02, respectively); the same finding occurred in younger patients with AML with intermediate karyotype (p = 0.008 and p = 0.01, respectively). In patients with intermediate karyotype, FLT3 internal tandem duplication (ITD) negatively affected EFS (EFS at 30 months: 30% vs. 6% in FLT3-ITD negative and FLT3 positive patients, respectively; p = 0.01) and OS (OS at 30 months: 38% vs. 20%, p = 0.03). The positive prognostic value of high WT1 expression does not have a clear explanation; it may be implicated either with WT1 anti-oncogenic function, or with the stimulating effect of WT1 oncogene on the leukemic cellular cycle, possibly associated with an enhanced response to chemotherapy.

Purpose: Treatment efficacy and toxicity are difficult to predict in lymphoma patients. In this study, the utility of circulating biomarkers in predicting and/or monitoring treatment efficacy/toxicity were investigated., Patients and Methods: Circulating biomarkers of cell death (nucleosomal DNA (nDNA) and cytokeratin 18 (CK18)), and circulating FLT3 ligand, a potential biomarker of myelosuppression, were assessed before and serially after standard chemotherapy in 49 patients with Hodgkin and non-Hodgkin lymphoma. Cytokeratin 18 is not expressed in lymphoma cells so is a potential biomarker of epithelial toxicity in this setting. Tumour response was assessed before and after completion of chemotherapy by 2D and 3D computed tomography radiological response., Results: Baseline nDNA level was significantly higher in all lymphoma subtypes compared with 61 healthy controls and was prognostic for progression-free survival in diffuse large B-cell lymphoma (DLBCL). Decreases in nDNA levels were observed in the first week after chemotherapy; in FL, early falls in nDNA predicted for long remission following therapy. In DLBCL, elevations in nDNA occurred in cases with progressive disease. Circulating CK18 increased within 48 h of chemotherapy and was significantly higher in patients experiencing epithelial toxicity graded >3 by Common Terminology for Classification of Adverse Events criteria. FLT3 ligand was elevated within 3-8 days of chemotherapy initiation and predicted those patients who subsequently developed neutropenic sepsis., Conclusion: These data suggest circulating biomarkers contribute useful information regarding tumour response and toxicity in patients receiving standard chemotherapy and have potential utility in the development of individualised treatment approaches in lymphoma. These biomarkers are now being tested within multicentre phase III trials to progress their qualification.

Objective: Hematopoietic stem cells are kept in a quiescent state in the hypoxic area of the bone marrow, which is essential for hematopoietic stem cell maintenance. However, when and how hematopoietic stem cells acquire their hypoxic state and maintain quiescence has not been fully elucidated. The aim of this study was to understand this process in human hematopoietic stem cells after bone marrow transplantation., Materials and Methods: Human CD34-positive cord blood cells were transplanted into nonobese diabetic/severe combined immunodeficient interleukin-2 receptor γ chain knockout mice. Cell cycle and hypoxia assay analyses were performed, to identify changes in the characteristics of human hematopoietic stem cells following transplantation. Quantitative real-time reverse transcription polymerase chain reaction analysis was used to analyze the transcriptional changes accompanying this transition., Results: Engrafted primitive lineage-negative CD34-positive CD38-negative cells acquired hypoxic state and quiescence in the recipient bone marrow between 4 and 8 weeks, and between 8 and 12 weeks after transplantation, respectively. During 4 and 8 weeks after transplantation, changes in the transcription levels of G₀ regulatory factors, such as CCNC and RBL1, and stem cell regulators, such as Flt3, were also seen, which may be related to the characteristic changes in the cell cycle or oxygenation state., Conclusions: Behavioral changes of hematopoietic stem cells in their cell cycle and oxygenation state during and after bone marrow engraftment may provide insights into hematopoietic stem cell regulation, mediating the improvement of clinical hematopoietic stem cell transplantation protocols and the eradication of leukemia stem cells., (Copyright © 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Acute myeloid leukemia (AML) is maintained by rare leukemia-initiating cells (L-ICs). FLT3 and/or PI3K pathways are often dysregulated in AML and may be important for L-IC survival. The presence of PI3K pathway intermediate integrin linked kinase (ILK), and FLT3 was confirmed in five L-IC-enriched AML patient samples. Treatment of AML cells with QLT0267, an inhibitor of ILK and FLT3, decreased survival of long-term suspension culture-initiating cells and NOD/SCID mouse L-IC. In contrast, little toxicity toward normal bone marrow progenitors was observed, demonstrating that candidate leukemic stem cells can be eliminated by inhibition of these targets while normal hematopoietic counterparts are spared., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

CD103(+) dendritic cells (DCs) in nonlymphoid tissues are specialized in the cross-presentation of cell-associated antigens. However, little is known about the mechanisms that regulate the development of these cells. We show that two populations of CD11c(+)MHCII(+) cells separated on the basis of CD103 and CD11b expression coexist in most nonlymphoid tissues with the exception of the lamina propria. CD103(+) DCs are related to lymphoid organ CD8(+) DCs in that they are derived exclusively from pre-DCs under the control of fms-like tyrosine kinase 3 (Flt3) ligand, inhibitor of DNA protein 2 (Id2), and IFN regulatory protein 8 (IRF8). In contrast, lamina propria CD103(+) DCs express CD11b and develop independently of Id2 and IRF8. The other population of CD11c(+)MHCII(+) cells in tissues, which is CD103(-)CD11b(+), is heterogenous and depends on both Flt3 and MCSF-R. Our results reveal that nonlymphoid tissue CD103(+) DCs and lymphoid organ CD8(+) DCs derive from the same precursor and follow a related differentiation program.

Impact of FLT3 receptor tyrosine kinase activation via internal tandem duplication (ITD) of the juxtamembrane region on outcome of acute myeloid leukemia (AML) is still controversial. Recent researches reveal a role of FLT3 in monocyte differentiation in hematopoiesis. We analyzed the clinical impact of FLT3 alterations in adult AML patients excluding acute promyelocytic leukemia (APL) who received induction chemotherapy according to morphologic classification. Retrospective review of medical records from three centers in Korea between 1997 and 2007 was performed. Polymerase chain reaction was performed on genomic DNA derived from blood samples of patients before induction chemotherapy for FLT3-ITD detection. We assessed overall survival (OS), first disease-free survival (1-DFS), and response to induction chemotherapy. One hundred eighty-four patients (median age 49.1 years, range 16.0-76.5) with AML excluding APL received induction chemotherapy from three centers. FLT3-ITD was detected in 22 patients. One hundred forty-one patients were below age 60. One hundred seventy-nine patients received induction chemotherapy with cytarabine and idarubicin (AId) regimen. One hundred nineteen patients achieved complete remission (CR) after first induction chemotherapy. FLT3-ITD was not related to achievement of CR. 1-DFS was longer in patients without FLT3-ITD (median 1-DFS 16.5 vs. 8.5 months, p = 0.025). 1-DFS was not different according to FLT3-ITD status in nonmonocyte lineage leukemia (p = 0.355), while 1-DFS was shorter in monocyte lineage leukemia for FLT3-ITD positive patients (20.9 vs. 2.4 months, p < 0.001). FLT3-ITD had no impact on OS except for monocyte lineage, where OS was significantly shorter in FLT3-ITD positive group (39.4 vs. 6.0 months, p = 0.026). Moreover FLT3-ITD was stronger prognostic factors in monocyte lineage AML than risk stratification based on cytogenetics. Status of FLT3-ITD should be analyzed differently in AML patients according to morphologic profile. FLT3-ITD is a predictive and prognostic marker only in monocyte lineage patients. This result suggests an existence of distinct subset of monocyte lineage AML with leukemogenesis involving FLT3 activating pathway.

There are several important biological markers in myeloid leukemia. In particular, WT1, FLT3, P-glycoprotein (P-gp) and surface antigens are important biologic markers. When we monitor the treatment of leukemia, WT1 is considered to be an important marker, and has been applied to immune therapy. The abnormality of the genes of FLT3 and expression of P-gp are poor prognostic factors, and their inhibitors are developed in progress. The surface antigen analysis of blast cells is also used in the classification and the treatment strategy decision. Recently, as for the treatment of leukemia, stratification by various kinds of factors has been adopted in many prospective studies. The results of these biological markers are utilized for the stratification.

Elderly acute myeloid leukaemia (AML) patients have a dismal prognosis due to biological features of disease in itself and to presence of comorbidities. Aim of this study was to evaluate the prognostic impact of comorbidity prognostic score systems applied in our population of patients. as well as other clinical-biological features. We retrospectively considered the outcome of 120 patients aged >65 years diagnosed as having AML between January 2001 and December 2005. Comorbidities were evaluated by using Charlson comorbidity index (CCI), Hematopoietic cell transplantation comorbidity index (HCTCI) and a score proposed by Dombret et al. in 2007. Median patient age was 67 years. Forty-six patients were treated with intensive chemotherapy and 23 reached a complete remission. Seventy-four patients received only supportive therapies or low-dose chemotherapy. Multivariate analysis showed the effects of leukocytosis (p = 0.0013), antecedent Myelodysplastic syndrome (MDS) (p = 0.011), FLT3 abnormalities (p = 0.032), CCI (p = 0.0037) and Dombret et al. score (p = 0.045) as independent prognostic parameters for survival. Based on these variables we were able to stratify patients in low and high risk, with different median overall survival: patients were considered as low risk if they had none or only one of the above mentioned adverse factors for survival, with a median overall survival of 447 days. Patients with two or more adverse factors were categorized as high risk: this subgroup had a median overall survival of 227 days (p = 0.001). Comorbidities are independent factors that influence survival. Application of CCI and Dombret score may help to better identify patients at diagnosis who can benefit from intensive chemotherapy.

A 35-year-old male with a FLT3(+) AML underwent allogeneic peripheral blood stem cell transplant using a myeloablative non-total body irradiation (TBI) conditioning regimen from his HLA-matched sibling donor. Following transplantation, he developed grade II acute graft-versus-host disease (GVHD) that resolved with increasing immunosuppression. The medications were subsequently discontinued, and he did not develop any evidence of chronic GVHD. Eighteen months after transplant, while off all immunosuppression, he developed fatigue and a blood count showed circulating blasts consistent with relapse of his disease. Among the various therapeutic questions is whether there is a role for a second allogeneic transplant to treat his disease and if so, at what time, with what conditioning, and with which type of donor.

Objective: CD34 is a sialomucin often expressed by cells with hemangiopoietic potential and widely serves as a surrogate marker of stem cell potential. Mesenchymal stromal cells (MSCs) also express CD34, although the functional significance of its expression remains undefined. In this study, we determined whether CD34(pos) MSCs are functionally distinct from CD34(null) MSCs., Materials and Methods: MSCs derived from C57Bl/6 mice were transduced to express the green fluorescent protein (GFP) from which pure CD34(pos) MSC and CD34(null) MSC clones were selected. In vitro, clones were examined by microarray analysis, while in vivo subcutaneous implantation of matrix-embedded MSCs was used to assess cell survival, differentiation, and neovascularization., Results: The flow cytometric phenotype of CD34(pos) and CD34(null) MSCs were similar, as was gene expression of vascular endothelial growth factors (VEGFs) A and B. However, CD34(pos) MSCs upregulated a number of supplementary angiogenesis-associated genes and showed a greater expression of gene associated with vascular differentiation. At 15 days postimplantation, cell survival between CD34(pos) and CD34(null) MSCs was similar, however, CD34(pos) MSCs evoked a significantly greater host-derived response (4.2 +/- 0.7 vs 1.9 +/- 0.5 x 10(6) cells; p < 0.05). GFP-expressing CD34(pos) MSC implants acquired significantly more CD31 expression compared to CD34(null) MSC cells (10.7% +/- 8.4% vs 3.1% +/- 0.6%; p < 0.05), as well as a significantly greater host-derived endothelial cell influx (CD31(+)/CD45(-))., Conclusion: CD34 expression by MSCs correlates with enhanced vasculogenic and angiogenic potential in vivo.

The thymus is seeded by bone marrow-derived progenitors that circulate in the blood. Multiple cell types can be found in the thymus early after i.v. administration or in steady state, but most fail to satisfy the known characteristics of true T progenitors. Cells that do conform to classical definitions retain multilineage potential, but surprisingly, cannot make B cells. Because acquisition of the T lineage fate among noncommitted progenitors is a lengthy process, the absence of B cell potential in early thymocytes suggests that B and T lineages diverge prethymically. To test this suggestion, we screened numerous presumptive progenitor populations for T cell growth and differentiation potential, as well as for clonogenic T or B cell development. We find that blood and marrow each contain multiple distinct subsets that display growth and differentiation potential consistent with being canonical T progenitors. Assessment of clonogenic potential further shows that although all blood and marrow populations have high T cell cloning potential, no T/non-B cells are apparent. These data suggest that either true thymic reconstitution potential derives from a small T/non-B cell subset of one of these populations, or that most of the cells defined as canonical progenitors within the thymus do not, in fact, reside in the mainstream of T progenitor differentiation.

Frequently direct measurement of proteins or their phosphorylation in intact cells is not possible, for instance, when cells are too few, frozen, or subject to degradation. We have demonstrated that tumor cells pour their DNA, RNA, and protein content into circulation because of turnover and breakdown of cell structures. Proteins in solution most likely circulate as complexes, which protects them from degradation. We describe a cell-free, bead-based method that takes advantage of this phenomenon. Our approach is based on immunoprecipitation of the protein of interest on the surface of beads, followed by detection of the protein or its modification (phosphorylation) using a secondary antibody labeled with phycoerythrin at a 1:1 ratio. Fms-like tyrosine kinase-3, which is mutated in majority of cases of acute myeloid leukemia, is used as an example. This method could be applied to the quantitation of several other proteins without the need for intact cells.

Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are present in approximately one third of patients with acute myeloid leukemia (AML) and are associated with adverse prognosis. The important role played by FLT3 in the survival and proliferation of blasts, and its overexpression in most patients with AML, make FLT3 an attractive therapeutic target. We undertook a phase 2 trial of the FLT3-selective tyrosine kinase inhibitor lestaurtinib (CEP701) used as monotherapy in untreated older patients with AML not considered fit for intensive chemotherapy, irrespective of FLT3 mutation status. Lestaurtinib was administered orally for 8 weeks, initially at a dose of 60 mg twice daily, escalating to 80 mg twice daily, and was generally well tolerated. Clinical activity, manifest as transient reductions in bone marrow and peripheral-blood blasts or longer periods of transfusion independence, was seen in 3 (60%) of 5 patients with mutated FLT3 and 5 (23%) of 22 evaluable wild-type FLT3 patients. Laboratory data demonstrated that clinical responses occurred where the presence of sustained FLT3-inhibitory drug levels were combined with in vitro cytotoxic sensitivity of blasts to lestaurtinib. Further evaluation of this compound, in combination with cytotoxic chemotherapy or other targeted agents, is warranted in both FLT3 mutant and wild-type patients.

Background: Vascular endothelial growth factor (VEGF) is a key regulator of physiologic as well as pathologic angiogenesis. The response of VEGF to endothelial cell mitogenesis and survival, as well as angiogenesis and microvascular permeability, is mainly mediated through its receptor-2, VEGFR2 (kinase domain receptor or fetal liver kinase-1, KDR or Flk-1). This study aimed to detect the expression of VEGFR2 in various forms of thyroid tumors. In addition, the expression of Flk-2 (receptor for Flt-3) and c-Kit (receptor for steel locus factor), which shows strong similarity to Flk-1, was also examined in thyroid tumors., Methods: RT-PCR analyses of c-Kit and immunohistochemical staining of c-Kit, Flk-1, and Flk-2 were performed in archived samples of 18 papillary thyroid carcinoma (PTC), 9 follicular thyroid carcinoma (FTC), 12 follicular adenoma (FA), and 7 nodular goiter (NG) samples. The data were correlated to clinicopathologic features., Results: By RT-PCR analyses, c-Kit expression was detected in 22% (4/18) of PTC, 22% (2/9) of FTC, 25% (3/12) of FA, and 57% (4/7) of NG samples. However, positive immunostaining signals of c-Kit were only observed in 17% (3/18) of PTC samples, and not in the others. Similarly, Flk-1 expression was only detected by immunohistochemistry in 67% (12/18) of PTC and 43% (3/7) of NG samples, and not in the others. Interestingly, the expression of Flk-2 was found in 89% (16/18) of PTC, 89% (8/9) of FTC, 75% (9/12) of FA, and 29% (2/7) of NG samples. An inverse relationship of thyroid cancer size with Flk-2 expression was found., Conclusion: Flk-2 expression was detected in various forms of thyroid tumors and increased Flk-2 expression was correlated with thyroid tumors with increased transforming activity, suggesting that Flk-2 is involved in pathogenic development of thyroid malignancy. Similarly, Flk-1 expression was also found in some thyroid tumors, while the expression of c-Kit-mediated pathways may not play a major role in thyroid tumorigenesis.

In vivo steady-state type I natural IFN-producing and dendritic cell (DC) development is largely dependent on Flt3 signaling. Natural IFN-producing and DC progenitors and their respective downstream cell populations express the flt3 receptor, and Flt3 ligand (Flt3L)(-/-) mice have reduced while Flt3L-injected mice develop markedly increased numbers of both cell types. In the present study, we show that SU11657, a small multitargeted receptor tyrosine kinase inhibitor with Flt3 affinity, suppressed in vitro natural IFN-producing and DC development in Flt3L-supplemented mouse whole bone marrow cell cultures in a dose-dependant manner, while DC development in GM-CSF-supplemented cultures was not affected. In vivo SU11657 application led to a significant decrease of both natural IFN-producing and DCs, comparable to the reduction observed in Flt3L(-/-) mice. Conversely, Flt3L plasma levels increased massively in inhibitor-treated animals, likely via a regulatory feedback loop, without being able to compensate for pharmacological Flt3 inhibition. No obvious toxicity was observed, and hemopoietic progenitor cell and stem cell function remained intact as assessed by myeloid colony-forming unit activity and in vivo bone marrow repopulation assays. Furthermore, upon treatment discontinuation, IFN-producing and DCs recovered to normal levels, proving that treatment effects were transient. Given the importance of IFN-producing and DCs in regulation of immune responses, these findings might lead to new pharmacological strategies in prevention and treatment of autoimmune diseases and complications of organ or blood cell transplantation.