Quantification of midostaurin in plasma and serum by stable isotope dilution liquid chromatography-tandem mass spectrometry: Application to a cohort of patients with acute myeloid leukemia.

OBJECTIVES: Midostaurin is an oral multitargeted tyrosine kinase inhibitor for the treatment of acute myeloid leukemia (AML). Therapeutic drug monitoring of midostaurin may support its safe use when suspecting toxicity or combined with strong CYP3A4 inhibitors. METHODS: A stable isotope dilution liquid chromatography-tandem mass spectrometry method was developed and validated for the determination and quantification of midostaurin in human plasma and serum. Midostaurin serum concentrations were analyzed in 12 patients with FMS-like tyrosine kinase 3 (FLT3)-mutated AML during induction chemotherapy with cytarabine, daunorubicin, and midostaurin. Posaconazole was used as prophylaxis of invasive fungal infections. RESULTS: Linear quantification of midostaurin was demonstrated across a concentration range of 0.01-8.00 mg/L. Inter- and intraday imprecisions of the proposed method were well within ±10%. Venous blood samples were taken in nine and three patients in the first and second cycle of induction chemotherapy. Median (range) midostaurin serum concentration was 7.9 mg/L (1.5-26.1 mg/L) as determined in 37 independent serum specimens. CONCLUSION: In a real-life cohort of AML patients, interindividual variability in midostaurin serum concentrations was high, highlighting issues concerning optimal drug dosing in AML patients. A personalized dosage approach may maximize the safety of midostaurin. Prospective studies and standardization of analytical methods to support such an approach are needed.

Phase 1 study of combinatorial sorafenib, G-CSF, and plerixafor treatment in relapsed/refractory, FLT3-ITD-mutated acute myelogenous leukemia patients.

Stroma-leukemia interactions mediated by CXCR4, CD44, VLA4, and their respective ligands contribute to therapy resistance in FLT3-ITD-mutated acute myelogenous leukemia (AML). We conducted a phase 1 study with the combination of sorafenib (a FLT3-ITD inhibitor), plerixafor (a SDF-1/CXCR4 inhibitor), and G-CSF (that cleaves SDF-1, CD44, and VLA4). Twenty-eight patients with relapsed/refractory FLT3-ITD-mutated AML were enrolled from December 2010 to December 2013 at three dose levels of sorafenib (400, 600, and 800 mg twice daily) and G-CSF and plerixafor were administered every other day for seven doses starting on day one. Sorafenib 800 mg twice daily was selected for the expansion phase. While no dose-limiting toxicities (DLT) were encountered in the four-week DLT window, hand-foot syndrome and rash were seen beyond the DLT window, which required dose reductions in most patients. The response rate was 36% (complete response (CR) = 4, complete remission with incomplete platelet recovery (CRp) = 4, complete remission with incomplete hematologic recovery (CRi) = 1, and partial response (PR) = 1) for the intention to treat population. Treatment resulted in 58.4 and 47 mean fold mobilization of blasts and CD34 /38- stem/progenitor cells, respectively, to the circulation. Expression of the adhesion molecules CXCR4, CD44, and VLA4 on circulating leukemia cells correlated negatively with the mobilization of CD34+/38-, CD34+/38-/123+ "progenitor" cells (all P ≤ .002). Mass cytometry analysis of sequential samples from two patients demonstrated resistance emerging early on from sub-clones with persistent Akt and/or ERK signaling. In conclusion, the strategy of combined inhibition of FLT3 kinase and stromal adhesive interactions has promising activity in relapsed/refractory, FLT3-ITD-mutated AML, which warrants further evaluation in the front-line setting.

Analysis of pre-chemotherapy WBC, PLT, monocyte, hemoglobin, and MPV levels in acute myeloid leukemia patients with WT1, FLT3, or NPM gene mutations.

Effects of mutations on AML (acute myeloid leukemia) patients have been an area of clinical interest. The aim of this study was to analyze pre-chemotherapy WBC (white blood cell), platelet, monocyte, hemoglobin, and mean platelet volume (MPV) levels in acute myeloid leukemia patients with Wilms tumor 1 (WT1), FMS-like tyrosine kinase 3 (FLT3), or nucleophosmin (NPM) gene mutations, attempting to detect and compare possible differences in these values.The study included 71 patients with acute myeloid leukemia known to have WT1, FLT3, or NPM gene mutations. The patients were divided into 3 groups: FLT3-mutated AML patients without any accompanying known mutations other than WT1 at the time of diagnosis (Group 1), NPM-mutated AML patients without any accompanying known mutations other than WT1 at the time of diagnosis (Group 2), WT1-mutated AML patients with no other accompanying known mutations at the time of diagnosis (Group 3). We carried out intergroup comparisons of WBC, platelet (PLT), monocyte, hemoglobin, and MPV levels before chemotherapy.There was a statistically significant difference between the groups in terms of WBC parameters (P = .001). There were no statistically significant differences between the groups with respect to hemoglobin, platelet, and monocyte levels.Higher white blood cell counts could be observed in patients with FLT3-mutated AML.

A Phase II Study of Midostaurin and 5-Azacitidine for Untreated Elderly and Unfit Patients With FLT3 Wild-type Acute Myelogenous Leukemia.

BACKGROUND: Midostaurin, a multikinase inhibitor, is approved for treatment of FLT3-mutant acute myeloid leukemia (AML). A phase I study established that midostaurin 75 mg orally twice daily for 14 days with standard dose azacitidine was safe and tolerable in elderly patients with AML. Herein, we report the phase II expansion cohort of previously untreated elderly or unfit patients with AML. PATIENTS AND METHODS: Primary objectives were to further describe the toxicity profile and determine the response rate in untreated patients with AML. Patients received midostaurin 75 mg orally twice daily on days 8 to 21 in combination with intravenous azacitidine at 75 mg/m2 on days 1 to 7. Plasma inhibitory activity assay for FLT3 was performed pretreatment and on day 8 and day 15 of each cycle. RESULTS: Twenty-six patients (median age, 74 years; range, 59-85 years) with FLT3 wild-type AML were accrued. Patients received a median of 2 cycles of therapy (range, 1-10 cycles). Seven (29%) of 24 evaluable patients achieved a clinical response (4 complete response; 1 complete response with incomplete count recovery; and 2 partial response). The median overall survival was 244 days (95% confidence interval, 203-467 days). Hematologic, infectious, and gastrointestinal toxicities were comparable to similar studies. Peripheral blood FLT3 wild-type phosphorylation declined to 8% to 55% of pretreatment by day 15 of cycle 1 (7 patients) and declined with subsequent cycles (< 10% baseline) in 2 patients who were analyzed after cycle 3. CONCLUSION: Multiple cycles of azacitidine and midostaurin were not well-tolerated, but persistent inhibition of FLT3 wild-type phosphorylation suggest intermittent dosing of midostaurin should be considered in future low-intensity regimens for FLT3-mutant AML.

Preliminary observation of chemokine expression in patients with Stanford type A aortic dissection.

Stanford type A Aortic dissection (TAAD) is a deadly cardiovascular disease but the relationship between inflammatory cytokines and disease pathogenesis is still unclear. Observation of the changes of different chemokines may help to explore the etiology of TAAD much further. Clinical data was collected from TAAD patients (TAAD group) and healthy controls (HC group) in our institute between October 2013 and December 2014. Blood sample was harvested from each subject of two groups. The expression levels of eighty chemokines were examined by protein array technology. Then we tested the expressions of macrophage inflammatory protein 1ß (MIP-1ß), epithelial neutrophil activating peptide 78 (ENA-78), interleukin 16 (IL-16), interferon inducible protein 10 (IP-10), and FMS-like tyrosine kinase 3 (Flt-3) ligand by using luminex technology. Osteopontin (OPN) and monocyte chemotaxis protein (MCP) levels were analyzed by ELISA kits. The mean age of TAAD group is 49.9 ±â€¯11.2 and 48.7 ±â€¯9.9 in HC group, respectively. 76.0% of TAAD patients and 72.0% of healthy controls were male. MIP-1ß and ENA-78 expression in TAAD group were significantly lower than that in HC group, while significant increasing IL-16 level was found. Plasma levels of OPN in TAAD group increased remarkably compared with HC group, but MCP-1 and MCP-2 expression significantly decreased. No correlation was shown between serum CRP levels and plasma level of these cytokines by using Spearman analysis. ROC analysis showed that OPN could be indicators for TAAD diagnosis with sensitivity of 0.92 and specificity of 0.99. Our results provide a reasonable way to focus on the chemokines in understanding the pathogenesis of human TAAD.

A quantitative method for detection of circulating fms related tyrosine kinase 3 (FLT-3) in acute myeloid leukemia (AML) patients.

FMS related tyrosine kinase 3 (FLT-3) is a tyrosine kinase expressed in early hematopoietic precursor cells and has roles in survival, proliferation, and differentiation. Bone marrow expression and mutagenic analysis of FLT-3 in Acute Myeloid Leukemia (AML) patients is well-characterized. However, the levels of circulating FLT-3 in serum have not been previously described. In this study we describe a quantitative electrochemiluminescent immunoassay that detects FLT-3 in human serum. Using this method we find that AML patients have elevated levels of circulating FLT-3 and these levels correlated to the percent blast counts in the bone marrow (BM).

Cre/lox Studies Identify Resident Macrophages as the Major Source of Circulating Coagulation Factor XIII-A.

OBJECTIVE: To establish the cellular source of plasma factor (F)XIII-A. APPROACH AND RESULTS: A novel mouse floxed for the F13a1 gene, FXIII-Aflox/flox (Flox), was crossed with myeloid- and platelet-cre-expressing mice, and cellular FXIII-A mRNA expression and plasma and platelet FXIII-A levels were measured. The platelet factor 4-cre.Flox cross abolished platelet FXIII-A and reduced plasma FXIII-A to 23±3% (P<0.001). However, the effect of platelet factor 4-cre on plasma FXIII-A was exerted outside of the megakaryocyte lineage because plasma FXIII-A was not reduced in the Mpl-/- mouse, despite marked thrombocytopenia. In support of this, platelet factor 4-cre depleted FXIII-A mRNA in brain, aorta, and heart of floxed mice, where FXIII-Apos cells were identified as macrophages as they costained with CD163. In the integrin αM-cre.Flox and the double copy lysozyme 2-cre.cre.Flox crosses, plasma FXIII-A was reduced to, respectively, 75±5% (P=0.003) and 30±7% (P<0.001), with no change in FXIII-A content per platelet, further consistent with a macrophage origin of plasma FXIII-A. The change in plasma FXIII-A levels across the various mouse genotypes mirrored the change in FXIII-A mRNA expression in aorta. Bone marrow transplantation of FXIII-A+/+ bone marrow into FXIII-A-/- mice both restored plasma FXIII-A to normal levels and replaced aortic and cardiac FXIII-A mRNA, while its transplantation into FXIII-A+/+ mice did not increase plasma FXIII-A levels, suggesting that a limited population of niches exists that support FXIII-A-releasing cells. CONCLUSIONS: This work suggests that resident macrophages maintain plasma FXIII-A and exclude the platelet lineage as a major contributor.

Standardized Centile Curves and Reference Intervals of Serum Thymidine Kinase 1 Levels in a Normal Chinese Population Using the LMS Method.

AIMS: The aim of the present study was to re-establish a set of normative data for serum thymidine kinase 1 (STK1) for the Chinese population. METHODS: The study included 14,960 Chinese subjects (9586 males and 5374 females) from 20 to 79 years old. Subjects suffering from diseases that could affect STK1 levels were excluded. STK1 was measured by a sensitive chemiluminescence dot blot assay. The reference intervals were calculated using the LMS method. RESULTS: Peak thymidine kinase 1 (TK1) serum levels were observed at 20 years of age for both genders. After the age of 20 years, serum TK1 levels decreased slowly from 0.51 to 0.36 pM, reaching a plateau to a mean of 0.35 pM in late adulthood. The mean pretreatment STK1 Z-scores in patients with solid malignant tumors obtained from related studies for cancers were 0.01 ± 0.99 (males, -0.07 ± 0.97; females, 0.09 ± 1.02). CONCLUSIONS: The present study established age- and gender-specific normative STK1 data for the Chinese population and showed the utility of these references for screening patients with solid malignant tumors.

Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia.

Owing to the more recent positive results with the anti-CD33 immunotoxin gemtuzumab ozogamicin, therapy against acute myeloid leukemias (AMLs) targeting CD33 holds many promises. Here, CD33 and CD123 expression on AML blasts was studied by flow cytometry in a cohort of 319 patients with detailed information on French-American-British/World Health Organization (FAB/WHO) classification, cytogenetics and molecular aberrations. AMLs of 87.8% express CD33 and would therefore be targetable with anti-CD33 therapies. Additionally, 9.4% of AMLs express CD123 without concomitant CD33 expression. Thus, nearly all AMLs could be either targeted via CD33 or CD123. Simultaneous presence of both antigens was observed in 69.5% of patients. Most importantly, even AMLs with adverse cytogenetics express CD33 and CD123 levels comparable to those with favorable and intermediate subtypes. Some patient groups with unfavorable alterations, such as FMS-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations, high FLT3-ITD mutant/wild-type ratios and monosomy 5 are even characterized by high expression of CD33 and CD123. In addition, blasts of patients with mutant nucleophosmin (NPM1) revealed significantly higher CD33 and CD123 expression pointing toward the possibility of minimal residual disease-guided interventions in mutated NPM1-positive AMLs. These results stimulate the development of novel concepts to redirect immune effector cells toward CD33- and CD123-expressing blasts using bi-specific antibodies or engineered T cells expressing chimeric antigen receptors.

A fast and simple approach for the simultaneous detection of hematopoietic chimerism, NPM1, and FLT3-ITD mutations after allogeneic stem cell transplantation.

Hematopoietic chimerism can be used as a tool for patient management after allogeneic hematopoietic stem cell transplantation (HSCT). An increase in the proportion of recipient cells after transplantation is strongly associated with relapse in chronic myeloid leukemia. However, in acute myeloid leukemia (AML) the significance of increasing mixed chimerism (MC) as a predictive marker for relapse is less clear. Several mutations frequently found in AML have been employed for minimal residual disease detection and relapse prediction. Therefore, a combined analysis of hematopoietic chimerism and of the molecular aberrations found in AML could be used to improve MC characterization. We developed a multiplex PCR for use in the simultaneous detection of hematopoietic chimerism and mutations in nucleophosmin (NPM1) and fms-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD). A total of 303 samples from 20 AML patients were analyzed after HSCT. The microsatellite markers used for hematopoietic chimerism detection were D1S80, D7S1517, D4S2366, THO1, and SE33. A total of 149 samples from 18 patients showed MC with a mean detection time of 9.7 months. From the 18 patients with MC, in 6 of the patients, no FLT3-ITD or NPM1 mutation was found at any time point tested, and these patients remained in complete hematological remission. In 12 patients with MC, FLT3-ITD and NPM1 mutations were found, and these patients showed signs of hematological relapse. Our combined analysis of NPM1/FLT3-ITD mutations and hematopoietic chimerism improved the characterization of patients with MC after HSCT. The present approach may be further expanded by combining additional mutations found in AML with hematopoietic chimerism detection.

Role of Fms-like tyrosine kinase 3 ligand as a potential biologic marker of lymphoma in primary Sjögren's syndrome.

OBJECTIVE: Patients with primary Sjögren's syndrome (SS) are at greater risk of developing lymphoma. This study was undertaken to evaluate whether the Fms-like tyrosine kinase 3 ligand (Flt-3L) might be associated with lymphoma in primary SS. METHODS: Serum levels of Flt-3L were measured in 369 patients with primary SS from the French Assessment of Systemic Signs and Evolution of Sjögren's Syndrome study cohort and in 10 patients with primary SS at the time of lymphoma diagnosis in an Italian cohort. Associations between increased levels of Flt-3L and a history of lymphoma, history of previously diagnosed criteria related to a high risk of lymphoma, and greater extent of disease activity were evaluated. RESULTS: Among patients with primary SS, higher levels of Flt-3L were significantly associated with a history of lymphoma (P = 0.0001). Previous markers for risk of lymphoma development, such as presence of purpura, low levels of C4, presence of lymphocytopenia, low levels of IgM, high levels of ß2 -microglobulin, and a higher primary SS disease activity score, were all associated with higher levels of Flt-3L. The levels of Flt-3L were also increased in serum obtained from patients with primary SS at the time of lymphoma diagnosis. Furthermore, the Flt-3L levels were elevated in the serum of 6 patients up to 94 months (mean 46 months) prior to the diagnosis of lymphoma. Receiver operating characteristic curve analysis showed that an Flt-3L level of 175 pg/ml was the ideal cutoff value for demonstrating an association with lymphoma (specificity 97.5%, sensitivity 44%, negative predictive value 97%). CONCLUSION: Flt-3L is associated with lymphoma in primary SS, and constitutes a good biologic marker. Higher levels of this cytokine are present several years before the diagnosis of lymphoma, and may be useful as a predictive marker of lymphoproliferative disorders in primary SS.

Mitigating effects of hUCB-MSCs on the hematopoietic syndrome resulting from total body irradiation.

This study evaluated the clinical and pathologic effects of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in the recovery from total body irradiation by comparing it with the effects of granulocyte-colony stimulating factor (G-CSF), an efficacious drug in the treatment of acute bone marrow radiation syndrome. BALB/c mice were treated with G-CSF or hUCB-MSCs after they were irradiated with 7 Gy cobalt-60 γ-rays. Circulating blood counts, histopathologic changes in the bone marrow, and plasma level of Flt-3L and transforming growth factor (TGF-ß1) were monitored in the postirradiation period. Hematologic analysis revealed that the peripheral leukocyte counts were markedly increased in the hUCB-MSCs-treated group, whereas G-CSF-treated mice did not recover significantly. Moreover, differential counts showed that hUCB-MSC treatment has regenerative effects on white blood cells, lymphocytes, and monocytes compared with the irradiated group. Treatment with hUCB-MSCs or G-CSF significantly increased immunoreactivity of Ki-67 until 3 weeks after total body irradiation. However, at 3 weeks, the number of Ki-67 immunoreactive cells significantly increased in the hUCB-MSCs-treated group compared with the G-CSF-treated group. Furthermore, hUCB-MSC treatment significantly modulated plasma levels of the hematopoietic cytokines Flt-3L and TGF-ß1, whereas G-CSF treatment failed to decrease the plasma Flt-3L levels at 2 weeks after irradiation. Based on the differences in circulating blood cell reconstitution and cell density of bone marrow, the authors suggest that MSC treatment is superior to G-CSF treatment for hematopoietic reconstitution following sublethal dose radiation exposure.

Molecular involvement and prognostic importance of fms-like tyrosine kinase 3 in acute myeloid leukemia.

AML (Acute myeloid leukemia) is a form of blood cancer where growth of myeloid cells occurs in the bone marrow. The prognosis is poor in general for many reasons. One is the presence of leukaemia-specific recognition markers such as FLT3 (fms-like tyrosine kinase 3). Another name of FLT3 is stem cell tyrosine kinase-1 (STK1), which is known to take part in proliferation, differentiation and apoptosis of hematopoietic cells, usually being present on haemopoietic progenitor cells in the bone marrow. FLT3 act as an independent prognostic factor for AML. Although a vast literature is available about the association of FLT3 with AML there still is a need of a brief up to date overview which draw a clear picture about this association and their effect on overall survival.

Assessment of circulating biomarkers for potential pharmacodynamic utility in patients with lymphoma.

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.

[Correlation of Fms-like tyrosine kinase 3 (FLT3) gene expression to FLT3/internal tandem duplication mutation in peripheral blood of acute myeloid leukemia.].

BACKGROUND AND OBJECTIVE: Fms-like tyrosine kinase 3 internal tandem duplication (FLT3/ITD) is associated with an unfavorable prognosis in acute myeloid leukemia (AML). However, the role of FLT3 expression as well as its correlation to FLT3/ITD has not sufficiently studied. This study was to evaluate the relationship between FLT3 gene expression and FLT3/ITD mutation in patients with de novo AML. METHODS: FLT3 gene expression was determined by real-time quantitative polymerase chain reaction (RQ-PCR). FLT3/ITD mutation was detected by PCR in 79 de novo AML patients. RESULTS: FLT3/ITD mutations were found in 22.8% (18/79) patients. FLT3 gene expression (range: 0-7320, median: 312) was detected in 92.4% (73/79) patients, but not in normal controls. Compared to AML patients with low FLT3 expressers and without FLT3/ITD mutation, patients with high FLT3 expressers and FLT3/ITD mutation had a significantly higher white blood count as well as a higher ratio of bone marrow blasts. The positive rate of FLT3/ITD mutation was not correlated to the level of FLT3 expression, and no statistical difference of FLT3 expression was found between AML patients with and without FLT3/ITD mutation. The complete remission (CR) rate of AML patients with FLT3/ITD mutation (58.8%) was significance lower than that of those without FLT3/ITD mutation (82.1%). In AML patients without FLT3/ITD mutation, the CR rate was significantly lower in patients with high FLT3 expressers (69.2%) than in those with low FLT3 expressers (93.3%). CONCLUSION: The FLT3 expression is not associated with FLT3/ITD mutation. High-FLT3 expression may be a poor prognostic factor for AML patients without FLT3/ITD mutation.

Plasma levels of FL and SDF-1 and expression of FLT-3 and CXCR4 on CD34+ cells assessed pre and post hematopoietic stem cell mobilization in patients with hematologic malignancies and in healthy donors.

Peripheral blood stem cells (PBSC) became the main source of cells for autologous transplantation. Alterations in the expression of adhesion molecules are essential in the CD34+ cells mobilization process. These molecules are involved in the interaction between hematopoietic and stromal cells and they have been disclosed as a considerable factor to the trafficking and homing of the CD34+ progenitor cells. This is a non-randomized PBSC mobilization study designed to evaluate the influence and behavior of FL and SDF-1 and their receptors in two different moments, prior and after HPCs mobilization, with the yield of CD34+ cells collected by apheresis. There was higher concentration of FL and lower of SDF-1 plasma level at post than pre PBSC mobilization (p=0.001 and p=0.012, respectively) regarding all individuals searched, but without any correlation with a good yield of CD34+ cells. However, CXCR4 expressions on the CD34+ cells from bone marrow aspirates (BMA), at pre and post mobilization showed a difference statistical significant for those individuals with good yield of CD34+ cells (p=0,036), but not achieved for poor yield (p=0,156). There was a higher expression of CXCR4 in steady-state for the successfully individuals than for those unsuccessfully (529.84+/-54.68 and 496.31+/-97.51, respectively). In conclusion, we confirmed the important role of CXCR4/SDF-1 axis in the process of PBSC mobilization.

Transcriptional signals of T-cell and corticosteroid-sensitive genes are associated with future acute cellular rejection in cardiac allografts.

BACKGROUND: Profiling mRNA levels of 11 informative genes expressed by circulating immune effector cells identifies cardiac allograft recipients at low risk for current moderate-severe acute cellular rejection (ACR). METHODS: We conducted a nested case-control study of 104 cardiac allograft recipients to investigate the association of transcriptional profiles of blood samples with either a future rejection episode within 12 weeks of a baseline clinical sample or persistent histologic quiescence for the same time period. RESULTS: The transcription profile yielded a score (0 to 40 scale) of 27.4 +/- 6.3 for future rejectors (n = 39) and 23.9 +/- 7.1 for controls (n = 65) (p = 0.01). In patients who were

Cell-free bead-based detection of total and phosphorylated proteins in plasma and cell lysates: detection of FLT3.

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.