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  • 1
    Online Resource
    Online Resource
    Prognostic Significance of FLT3 and NPM1 Mutations in Adults of Age 18–60 with De Novo Acute Myeloid Leukemia (AML) on SWOG S0106 Study: A Study by FHCRC and SWOG
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 2520-2520
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 2520-2520
    Abstract: Abstract 2520 INTRODUCTION. Age, cytogenetics, FLT3 and NPM1 mutations are the most significant prognostic factors (PFs) for adult AML treated with standard regimens, but the predictive significance of FLT3 and NPM1 with contemporary treatments is unknown. We examined the clinical significance of NPM1 and FLT3 mutations in adult de novo AML pts enrolled on SWOG study S0106. METHODS. S0106 was a randomized phase III clinical trial for pts of age 18–60 with de novo non-M3 AML, evaluating the effects of adding Gemtuzumab Ozogamicin (GO) to standard induction therapy (Cytosine Arabinoside and Daunomycin, AD), and of post-consolidation GO vs. no additional therapy (ASH, 2009, Abstract 790). Samples from 198 of the 600 eligible pts were evaluated. Analyses for nucleotide insertions in exon 12 of the NPM1 gene and internal tandem duplications (ITD) within exons 14–15 of FLT3 were performed using fragment analyses in diagnostic bone marrow (BM, N=190) and peripheral blood (PB, N=8) samples. Mutant/wild-type (WT) allelic ratios (AR) were computed for all mutations. Effects of mutations and other PFs on complete response (CR), resistant disease (RD), overall survival (OS) and relapse-free survival (RFS) were analyzed by logistic and Cox regression. P-values are 2-sided. RESULTS. Patient characteristics and outcomes are shown in Table 1. In univariate analyses, NPM1-Mut pts had significantly higher CR (81% vs. 58%, P=.0018) and lower RD (13% vs. 28%, P=.028) rates, better OS (64% vs. 47%, P=.045) and RFS (54% vs. 41%, P=.50). FLT3-ITD was not associated with CR or RD, but was associated with poorer OS (hazard ratio [HR] 2.28, P=.0011) and RFS (HR 2.74, P=.0009). FLT3-ITD length (range 18–366, median 46), FLT3 AR (range 0.18–8.2, median 0.98), and NPM1 AR (range 0.2–1.0, median 0.8) were not associated with CR, RD, or OS, but RFS tended to be lower with higher ITD length (P=.076). In multivariate analyses with other PFs, neither NPM1 nor FLT3 was associated with CR or RD rates, however the combined effects of FLT3 and NPM1 identified 3 mutation risk groups for OS (P=.0044, Fig 1A) and RFS (P=.0003, Fig 1B), since NPM1 did not significantly affect outcomes within the FLT3-ITD pts. These risk groups are FLT3-WT/NPM1-Mut (Good Risk: 3-yr OS 82%, RFS 69%), FLT3-WT/NPM1-WT (Intermediate Risk: OS 49%, RFS 43%), and FLT3-ITD (Poor Risk: OS 29%, RFS 14%). The impact of adding GO to induction therapy was examined within each risk group. In each risk group, CR rates were higher in the AD+GO arm, though not significantly so. Likewise, the RD rates were lower in the AD+GO arm, but this difference was significant only in the largest group: Intermediate Risk, FLT3-WT/NPM1-WT, 17% vs. 34% (P=.026). Treatment arm did not significantly affect OS and RFS in any mutation risk group. CONCLUSION. This study confirmed prognostic effects of FLT3 and NPM1 mutations in de novo AML pts treated with AD or AD+GO. Analyses of the joint impact of NPM1 and FLT3 mutations do not rule out the possibility that they act independently. With the small numbers of pts in the “good” and “poor” risk groups, there was no clear evidence that mutation status predicts clinical benefit from adding GO to therapy. We are evaluating additional samples and will update these results as data matures. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.2520.2520
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
    detail.hit.zdb_id: 1468538-3
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  • 2
    Online Resource
    Online Resource
    DNMT3A Mutations Independently Predict Poor Outcome in Older AML Patients: A SWOG Report,
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 3519-3519
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 3519-3519
    Abstract: Abstract 3519 Background: Mutations of the DNA methyltransferase 3A (DNMT3A) gene, which encodes DNA cytosine-5-methyltransferase 3A, have recently been reported to have prognostic implications in adult acute myeloid leukemia (AML). We studied the incidence and prognostic impact of these mutations, in the context of other prognostic markers, in older patients with de novo AML. Methods: Diagnostic specimens were available for 268 patients who were registered on SWOG trials S-9031 and S-9333 (both of which enrolled patients 〉 55 years), and S-9500 (which enrolled patients from 18–55 years). The median age of patients was 63 years (range, 18 to 88 years). The samples were analyzed for the presence of DNMT3A mutations via direct sequencing of exon 23. DNMT3A mutation status was correlated with clinical characteristics, other genetic risk factors (cytogenetics and mutations in NPM1, FLT3-ITD, Kit, CEBPA and IDH1/2) and outcome. Results: DNMT3A mutations were found in 50 patients (19%). A total of 9 different mutations were identified (R882H, R882C, R882S, L901R, C911Y, N879D, P904L, R887I and W893S). The codon most frequently mutated was R882 (n=44). There were no statistically significant differences in the median age, WBC, blast or platelet count at diagnosis between mutated DNMT3A and wild type (WT). The DNMT3A mutations occurred most commonly in cytogenetically normal (CN) patients and did not occur in those with core binding factor abnormalities. DNMT3A mutations were more common in NPM1+ patients (67% vs 27%, p 〈 0.01) and were somewhat more common in FLT-ITD+ patients (26% vs 16%, p=0.059) patients. There were no differences in the incidence of CEBPA, KIT, or IDH1/2 mutations among patients with DNMT3A mutations as compared to DNMT3A WT. When adjusting for age, PS, WBC, blast count, FLT3/NPM1 status, and cytogentics, the presence of DNMT3A mutations independently predicted worse OS (20% vs 29% at 2 years, HR=2.3, 95% CI: 1.4–3.9, p=0.002) and worse EFS (14% vs 23% at 2 years, HR=2,1, 95% CI: 1.3–3.3, p=0.003). In CN-AML, 33% of the patients (p 〈 0.001) harbored DNMT3A mutations. In this patient subgroup, multivariate analysis revealed that the DNMT3A mutations predicted a shorter OS (23% vs 40%, HR=1.8, 95% CI: 1.1–3.0, p=0.03) and EFS (17% vs 32%, HR=2.0, 95% CI: 1.2–3.2, p=0.01). Among the high-risk NPM1/FLT3-ITD subgroup (FLT3-ITD+ regardless of NPM status, and FLT3-ITD-/NPM1-) OS was significantly worse in the DNMT3A+ patients (12% vs 30%, HR=2.1, 95% CI=1.3–3.4, p=0.002) as was the EFS (12% vs 22%, HR=1.9, 95% CI= 1.2–2.9, p=0.006). Conversely, in the FLT3-ITD-/NPM1+ favorable subgroup, DNMT3A+ patients trended towards improved RFS (71% vs. 47%, HR=0.2, 95% CI=0.1–1.1, p=0.063), although there was no difference in OS (40% vs. 40%, HR=0.9, CI=0.4–2, p=0.82). When restricting analysis to elderly patients, DNMT3A+ patients older than 60 years had significantly worse EFS (0% vs 14%, HR=1.6, 95% CI: 1.0–2.3, p=0.04) and trended towards worse OS (10% vs 18%, HR=1.8, 95% CI: 1–1.2, p=0.08), although this did not reach statistical significance. Conclusions: The high prevalence of DNMT3A mutations and its independent negative impact on OS and EFS identifies DNMT3A as a potentially important molecular marker for patients with AML, especially those with CN-AML. Within the high-risk FLT3-ITD/ NPM1 group, DNMT3A mutations identify a subset of patients with especially poor prognosis. Our results are in keeping with the recent published studies on the prognostic impact of DNMT3A in patients with AML and add valuable information for risk stratification of older AML patients. DNMT3A mutation status should be evaluated prospectively in future adult AML clinical trials. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.3519.3519
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
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    detail.hit.zdb_id: 80069-7
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  • 3
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    Multidimensional Flow Cytometry Significantly Improves Upon the Morphologic Assessment of Post-Induction Marrow Remission Status – Comparison of Morphology and Multidimensional Flow Cytometry; A Report From the Children's Oncology Group AML Protocol AAML0531
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 939-939
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 939-939
    Abstract: Abstract 939 Initial response to induction chemotherapy is a significant predictor of outcome in leukemias, where those with rapid response have an improved outcome compared to the non-responders. Morphologic evaluation of post induction marrow has been the gold standard in assessing remission status in leukemias, where those with 〈 5% blasts are considered to be in complete remission (CR), whereas those with '5% blast are considered induction failures. MRC AML 10 study, using morphologic assessment of remission, delineated a clinically significant threshold of 15% marrow blasts after induction I, where those with 5–15% blasts were shown to have a similar outcome to those in morphologic CR, while those with 〉 15% blasts were considered to be high risk. The Children's Oncology Group (COG) phase III AML protocol AAML0531 which treated 1022 eligible patients without Down Syndrome (DS) on an MRC based chemotherapy backbone, utilized the 〉 15% post induction morphologic blast threshold, as assessed at the institutional level, to allocate patients to specific risk groups based on the observed post induction 1 blast prevalence. In this study, all patients regardless of initial response would receive a second course of similar induction chemotherapy. Those with 5–15% blasts after course 1 with no other risk features who achieved a CR after the second course were considered intermediate risk, but those with 〉 15% post course 1 blasts who achieved a CR after course 2 were considered high risk and allocated to stem cell transplantation from the most suitable donor in first CR. As part of this trial, multidimensional flow cytometry (MDF) was used to assess marrow response after each course of induction chemotherapy. As morphologic evaluation would not be able to distinguish normal and malignant blasts in the marrow, we inquired whether morphologic CR status correlates with MDF findings. Of the 1022 eligible non-DS patients treated on AAML0531, 784 patients had consented to the correlative biology studies and had available MDF data for correlation with morphology. Of the 784 patients, 185 patients (24%) had failed to achieve a morphologic CR ( 〉 5% blast by morphology) after the initial course of chemotherapy, of which 94 were partial remissions (PR, 5–15% blast) and 91 were persistent disease (PD, 〉 15%). Of these 185 patients who failed to achieve a morphologic CR, 67 patients (36%) had no evidence of disease by MDF, and the remaining 64% had MDF detectable disease. Clinical outcome evaluation of these patients who failed induction ( 〉 5% blast) at the end of course 1 based on MDF status demonstrated that disease free survival for those with MDF detectable disease was 24% compared to that of 52% in those with induction failure with no MDF detectable disease (p 〈 0.0001). Corresponding Overall survival was 48% and 76%, respectively (p=0.005). As 15% blast threshold at the end of induction 1 was used as a threshold for risk allocation, we specifically evaluated the prevalence and clinical implications of MDF-detectable disease in this cohort of high risk patients. Of the 91 patients with 〉 15% blast after course I, 25 (27%) had no evidence of disease by MDF. Disease-free survival at 3 years from end of induction in this high risk cohort with and without MDF-detectable disease was 20% and 55%, respectively (p 〈 0.0001) with a corresponding OS of 47% and 83% (p=0.014). This study highlights the fact that morphologic evaluation of marrow specimens may not be adequate for post induction disease assessment. Multi-dimensional flow cytometry provides significant information for accurate assessment of response. Disclosures: Smith: Seattle Genetics:; Eisai:; Archimedes Pharma: Membership on an entity's Board of Directors or advisory committees; Pfizer, Inc.: Membership on an entity's Board of Directors or advisory committees.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.939.939
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
    detail.hit.zdb_id: 1468538-3
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  • 4
    Online Resource
    Online Resource
    The Effect of Traumatic Diagnostic Lumbar Puncture in De Novo Pediatric Acute Myeloid Leukemia - a Report from the Children's Oncology Group
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 4016-4016
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 4016-4016
    Abstract: Background - In many adult protocols for acute myeloid leukemia (AML), the standard of care is to perform a lumbar puncture (LP) at the time of diagnosis only if there are central nervous system (CNS) symptoms present, and if absent, the LP is performed after a period of systemic therapy in order to avoid contamination of the cerebrospinal fluid (CSF) with circulating blasts in the case of a traumatic LP, or is not done at all. This differs from the practice in pediatric AML protocols where LP is performed as part of the diagnostic workup and intrathecal (IT) cytarabine given at the time of the diagnostic LP, as well as during therapy, similar to the pediatric ALL approach. We sought to determine the effect of an initial traumatic LP in a large cohort of pediatric patients with de novo AML. Methods - Treatment protocols COG AAML03P1 and AAML0531 enrolled 1344 patients with diagnostic LP and outcome data available. In these protocols, patients identified to be CNS2 (0-5 WBC with blasts present) or CNS3 ( 〉 5 WBC with blasts or CNS symptoms at diagnosis) prior to systemic therapy received additional IT therapy, while CNS1 (no blasts) patients received IT therapy only at the beginning of most courses of chemotherapy. Patients were analyzed for CNS status as well as diagnostic LP red cell count. A diagnostic LP with greater than 100 red blood cells was considered traumatic. The effect of traumatic LP on outcome was analyzed. Results - Among these patients, 949 were CNS1, 217 were CNS2 and 178 were CNS3. In looking at percentage of patients with more than 100 RBC in the CSF, there were 52 (5.48%) CNS1, 12 (5.53%) CNS2 and 55 (30.9%) CNS3 (three group comparison, p 〈 0.001). As well, in patients with 6 to 100 RBC in the CSF in each CNS group there was a significant difference with 150 (15.81%) CNS1, 64 (29.49%) CNS2 and 45 (25.28%) CNS3 patients, (p 〈 0.001). Figure 1 shows the number of patients in each CNS group with number of RBC in the diagnostic LP. The number of RBC in the CSF did not correlate with the degree of peripheral blood hyperleukocytosis. The outcomes of patients with traumatic initial LP showed that patients who had a traumatic initial LP did not have a significantly different OS (HR 1.14, p=0.405) or EFS (HR 1.22, p=0.132) from study entry, nor RR (HR 1.06, p=0.769) or TRM (HR 1.30, p=0.510) compared to those with an atraumatic LP. This was similarly seen in the subgroups, CNS1, CNS2, and CNS3. In examining patients with 0 RBC in their initial CSF, OS was similar among the CNS1, CNS2 and CNS3 patients (62.6%, 62.7% and 66.4% respectively, p=0.974). The OS was also similar for patients with 〈 100 RBC (64.5% ± 2.8%) and 〉 100 RBC (63.0% ±9.1%). In examining outcomes of CNS3 patients with a traumatic tap, there were no significant differences in OS, EFS, or RR among those with traumatic tap and those without. Overall though, multivariable analyses showed that CNS3 patients had significantly worse EFS from study entry compared to CNS1 and CNS2 patients (HR 1.58, p 〈 0.001) due to a higher RR (HR 1.64, p=0.003). The CNS3 patients with traumatic LP had similar OS and EFS compared to those without traumatic LP (OS 63.5% ± 15.2% vs 60.8% ± 11.4% respectively, and EFS 43.4% ± 15.3% vs 35.5% ± 10.9% respectively). Conclusion - There were significantly more CNS3 patients with 〉 100 RBC in the CSF than in the other 2 CNS groups. Outcome for these patients with traumatic LP vs those with atraumatic LP however were not different for EFS, OS or RR for all 3 CNS groups. It is likely that more than half of the CNS3 patients received extra IT therapy due to traumatic LP. In the majority of cases these IT therapies were given with sedation, potentially unnecessarily. The concern though that a traumatic initial LP contributes to worse outcome in CNS3 patients is not warranted. Delaying initial LP to align with adult practice, when peripheral blasts are cleared, would not change prognosis, but may lessen the number of patients who need additional IT therapy from traumatic LPs, thus sparing a significant proportion of children unnecessary procedures and CNS directed chemotherapy. Figure 1 Number of Red Blood Cells in the CSF, CNS Status and Number of Patients in Each Grouping Figure 1. Number of Red Blood Cells in the CSF, CNS Status and Number of Patients in Each Grouping Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.4016.4016
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
    detail.hit.zdb_id: 1468538-3
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  • 5
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    The Role of Notch in Vascular Endothelial Cell-Mediated Protection of AML Precursors from Targeted Therapy
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 2750-2750
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 2750-2750
    Abstract: Despite advances in the treatment of acute myeloid leukemia (AML), disease relapse following an initial period of remission remains a significant risk, suggesting the existence of resistant leukemic precursors. Recent studies indicate resistance may be imparted to the leukemic precursors by the hematopoietic niche within the bone marrow. Previously, we have shown that a key component of the niche, endothelial cells (EC), can support long-term growth of AML precursors in culture. Here, we show that Notch is a novel target to disrupt the protective AML-EC interactions and enhance treatment effectiveness. In preliminary studies of AML with FLT3 mutations, we demonstrated that leukemic precursors from primary human AML patient samples are protected from treatment with a small molecule FLT3 inhibitor, AC220, when cultured in the presence of EC. We assayed cell viability of AML cells following 3 days of treatment with AC220 (100nM) or control (DMSO) in EC co-culture or liquid culture, and found that cells survived AC220 treatment better in EC co-culture compared to liquid culture (average percent cell viability from 3 primary FLT3-ITD+ AML patient samples relative to DMSO control: liquid culture (3 ± 3%) vs. EC co-culture (35 ± 19%), p=0.04). To identify genes involved in conferring protection to AML cells, we performed genome-wide transcriptional analysis of AML cells following 2 days of treatment with AC220 or DMSO in EC co-culture. EdgeR was used to assess differences in gene enrichment across cell populations. We found 1171 and 555 genes were increased and decreased, respectively, in AC220-treated population compared to DMSO control-treated population AML based on a 2-fold change and FDR 〈 0.01. Amongst the differentially expressed genes, we found an enrichment of genes involved in Notch signaling pathway, including HES1, CDKN1A, and CCND1, in AML cells that survived treatment with AC220 compared to control. To evaluate a role for Notch signaling in EC-mediated growth of AML cells, we incubated AML cells in EC co-culture with inhibitory antibodies against Notch 1 and 2 receptors or control antibody for 2 weeks, and assayed the cells for colony-forming cell (CFC) activity. We found that inhibition of Notch signaling led to an increase in CFC formation. In contrast, when we combined AC220 with Notch 1 and 2 blocking antibodies in AML/EC co-culture, we found that inhibition of Notch signaling significantly reduced the number of total CFC and FLT3-ITD+ CFC compared to AC220 treatment combined with control antibody (p 〈 0.005). These results suggest a role for Notch in EC-mediated protection of resistant AML precursors and thus offer a potential therapeutic strategy for sensitizing resistant precursors to targeted therapies. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.2750.2750
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
    detail.hit.zdb_id: 1468538-3
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  • 6
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    Discovery and Validation of Cell-Surface Protein Mesothelin (MSLN) As a Novel Therapeutic Target in AML: Results from the COG/NCI Target AML Initiative
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 2873-2873
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 2873-2873
    Abstract: In efforts to discover genes uniquely expressed in childhood AML, we performed transcriptomesequencing (RNA-Seq) in pediatric AML and contrasted the expression signature to that in normal marrow hematopoiesis. This effort led to the discovery of over 200 genes that lack expression in normal hematopoietic cells, but are variably expressed in pediatric AML cells. Mesothelin(MSLN) was discovered to be one of the most highly expressed genes in a subset of childhood AML cases (p 〈 10-15).Mesothelin is a cell-surface protein that is expressed onmesothelial cells ofserosal lining. MSLN is over-expressed on a variety of solid tumors, including lung, pancreatic, and ovarian cancers, and is associated with increased malignant transformation, cellular proliferation, and tumor aggressiveness. Given its cell surface expression, MSLN has emerged as an attractive target for immunotherapeutic interventions in solid tumors in adults. In this study, RNA obtained from diagnostic bone marrow specimens from childhood AML (N=434) and normal marrow (N=20) was subjected to wholetranscriptomesequencing and MSLN expression was quantified and normalized and reported as reads perkilobaseof exon per million reads mapped (RPKM). Similar data was obtained from adult TCGA AML database. Quantitative RT-PCR (qRT-PCR) and multidimensional flowcytometry(MDF) was used for confirmation of the transcript and cell surface protein expression. TARGET AML methylation data was used for correlation with transcript expression. Of the 434 specimens analyzed, MSLN mRNA expression was variably expressed (RPKM range 0-618.8), with expression detected in 119 patients (27%). Confirmatory studies by qRT-PCR on specimens with and without MSLN expression (N=137) showed correlation between RNA-Seqand PCR data. Cell surface MSLN expression was assessed by MDF using a PE-conjugated MSLN antibody (PE-mesoAb) and verified expression of MSLN protein on the leukemic cell surface in every case with MSLN transcript expression (Figure 1A). Evaluation of CD34+/CD38- hematopoietic progenitor cells by PE-mesoAbdemonstrated lack of MSLN expression by MDF. Evaluation of matched diagnostic and relapse specimens from MSLN-expressing patients (n=27) confirmed that MSLN expression was largely stable (R2=0.87), thus substantiating its expression in the major AML clone. Comparison of MSLN expression in pediatric vs. adult AML demonstrated a higher prevalence in pediatric AML (TARGET: 27% vs. TCGA: 11%)(Figure 1B). Evaluation of the clinical and biologic features in MSLN expressing (MSLN+) and non-MSLN expressing (MSLN-) pediatric patients revealed that MSLN expression was rarely observed in patients with normal karyotype (p 〈 0.001) or with the most common somatic mutations of FLT3/ITD, NPM1, CEBPA (p 〈 0.001 in all cases). However, MSLN expression was significantly higher in patients with inv(16), t(8;21) and MLL translocations (p 〈 0.001, p 〈 0.001, and p=0.02 respectively; Figure 1C). Given that a majority of patients with core binding factor (CBF) AML were MSLN+, we evaluated the clinical implications of MSLN expression in this favorable risk cohort. Among CBF patients, MLSN+ patients (n=95) had a relapse risk of 51% vs. 32% in the MSLN- (n=62) cohort (p=0.03; Figure 1D), with a corresponding disease free survival of 46% vs. 64% respectively (p=0.03). We further inquired about the mechanism by which MSLN expression might be regulated. Whole genome sequencing data failed to identify any genomic alterations in MSLN that could result in high expression. Therefore, we interrogated the possibility of epigenetic regulation of MSLN expression. Integration of the expression and methylation profiling cases with matching RNA-Seqand methylation data (N=246) demonstrated thathypomethylationof the MSLN promoter significantly correlated with high MSLN expression, implicating epigenetic regulation in the expression of MSLN in AML. Mesothelin, a therapeutic target in solid tumors, is highly expressed in biologically distinct subsets of childhood AML. High expression on leukemic blasts and lack of expression in normal hematopoiesis makes this antigen an ideal target for therapeutic intervention in AML. As a cell surface protein, this antigen avails itself for immune targeting by antibody drug conjugates, CAR-T cells, and T cell receptor mediated targeting. Figure 1 Mesothelin expression in childhood AML. Figure 1. Mesothelin expression in childhood AML. Disclosures Loken: Hematologics: Employment, Equity Ownership. Pardo:Hematologics, Inc: Employment.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.2873.2873
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 7
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    CD33 Splicing Polymorphism Is a Strong Predictor of Therapeutic Efficacy of Gemtuzumab Ozogamicin in De Novo AML: Report from COG-AAML0531 Study
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 2743-2743
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 2743-2743
    Abstract: Gemtuzumab ozogamicin (GO), a CD33-targeted immunoconjugate, is a re-emerging as therapeutic for AML. We have previously discovered polymorphisms in CD33 coding region that might be associated with outcome in patients treated with GO. One particular coding polymorphism, CD33-SNP rs12459419-C 〉 T (Ala14Val), is located within the splice enhancer region of exon-2, leading to expression of an alternate splice isoform lacking exon-2. This alternate splice isoform (D2-CD33), would encode a protein product lacking the IgV domain, which is the binding site for GO and most if not all CD33 antibodies used for diagnostic immunophenotyping. We therefore hypothesized that the SNP rs12459419 genotype would be associated with differential expression of the D2-CD33 transcript levels and differential cell surface CD33 expression leading to genotype determined differential response to GO. We evaluated the genotype frequency and functional significance of rs12459419, its association with CD33 cell-surface expression on leukemic blasts, and clinical response in 816 children and young adults with AML randomized to GO vs. No-GO in the COG trial AAML0531. The CD33 SNP rs12459419 genotype frequency was-CC=51%, CT=39% and TT=10% in patients, similar to the observed frequency in the general population. Correlation of SNP allele frequency with CD33 transcript levels and surface CD33 expression (as determined by p67.6 antibody) demonstrated that the T-allele was significantly associated with higher levels of D2-CD33 transcript (P=4.7e-11, Figure 1) and with lower diagnostic leukemic cell CD33 surface intensity (P=1.93e-29). Clinical outcome based on the SNP genotype demonstrated that patients with CC-genotype had significantly lower RR of 26%±7% in the GO arm whereas those in the No-GO arm had a RR of 49%±9% (HR=0.468, P 〈 0.001). The corresponding DFS for CC genotype patients in the GO and No-GO arms was 65%±7% and 46%±9%, respectively (HR=0.597, P=0.004; Figure 1). In contrast, in those with heterozygous CT or homozygous TT genotype, GO exposure provided no clinical benefit in RR (CT: 38%±9% vs. 37±10%, P=0.975; TT: 46%±20% vs. 46%±20%, P=0.798) nor DFS (CT: 56%±9% vs. 60%±10% GO vs. No-GO, P=0.821; TT: 51%±20% vs. 54%±18%, GO vs. No-GO, P=0.972, Figure 1). We further evaluated the impact of the CD33 genotype on the efficacy of GO in different risk groups as well as in high vs. low CD33 expression cohorts. Patients in the low-risk (LR) group with the CC genotype treated with GO had a RR of 10%±8% vs. 37%±13% (P 〈 0.001) from remission. Standard-risk patients with CC genotype had a RR of 41±12% vs. 59±12% (P=0.056) and high-risk patients had a RR of 36%±27% vs. 70%±32% (P=0.073) for the GO and No-GO arms. In contrast there was no benefit of addition of GO in patients with the CT or TT genotypes within each risk group. Since CD33 expression has been recently associated with GO efficacy, we evaluated the association of rs12459419 genotypes in patients with low (N=153) and high CD33 (N=436) cell surface expression quartile-1 and quartiles-2-4, respectively. Significant improvement in RR was observed in patients with the CC genotype in the GO arm over those in the No-GO arm (P=0.001) within quartiles 2-4, but not with the CT/TT genotype (P=0.112). Within lower CD33 expression (quartile-1) cohort, a similar trend towards improvement in RR compared with those in the No-GO arm (P=0.055) was observed although the CC genotype was less frequent. Multivariate cox regression analysis that included genotype, risk status, and CD33 expression demonstrated that CD33 CC genotype was an independent predictor of response to GO (HR= 0.45, P 〈 0.001 for RR and HR=0.57, P=0.003 for DFS). The rs12459419 genotype mediates expression of the GO binding site and informs on which patients should receive GO. The knowledge of CD33 genotype and prediction of response to GO provides opportunities to use patient genotypes for selecting CD33-targeted therapies. Given only half of the patients are expected to have a response to GO, we propose that all prior GO containing studies be re-evaluated for response based on patient CD33 genotype. The efficacy of GO in patients with an appropriate antibody binding domain also raises the possibility of developing next-generation CD33 immunoconjugates with epitopes targeted to regions not affected by alternative splicing and SNPs. Disclosures Loken: Hematologics: Employment, Equity Ownership.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.2743.2743
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
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  • 8
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    Prevalence and Prognostic Significance of KIT Mutations in Pediatric Core Binding Factor AML Patients Treated with Gemtuzumab Ozogamicin: Results from the Randomized Phase III Children's Oncology Group Trial AAML0531
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 800-800
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 800-800
    Abstract: Mutations of KIT (KIT +) occur in children and adults with core binding factor (CBF) acute myeloid leukemia (AML) and cluster within exons 8 and 17. We previously reported a 19% prevalence of KIT mutations in pediatric CBF AML and lack of prognostic significance in serial pediatric cooperative trials. We also determined that gemtuzumab ozogamicin (GO) improves outcomes for a subset of CBF AML patients with higher CD33 expression enrolled on AAML0531, a randomized trial of conventional chemotherapy with or without GO. Thus, in this study, we determined whether the clinical outcome of patients with KIT + CBF AML is affected by GO treatment. COG AAML0531 enrolled 1022 eligible pediatric de novo AML patients of which 247 had CBF AML [137 t(8;21) and 110 inv(16)/t(16;16)] based on central cytogenetic review. Of these 247 patients, 218 had evaluable samples for KIT mutational analysis. Analysis included PCR amplification of exons 8 and 17 and fragment length analysis and direct sequencing to identify all missense and size mutations. Mutations were detected in 55 patient samples (25%); 27 (49%) involved exon 8, 26 (47%) involved exon 17 and 2 (4%) involved both exons. Breakdown by exon and CBF translocation type demonstrated exon 8 mutations in 12/121 (10%) t(8;21) samples and 17/97 (18%) inv(16)/t(16;16)patient samples. Exon 17 mutations were found in 18/121 (15%) t(8;21) and 10/97 (10%) inv(16)/t(16;16) patient samples. Overall outcome analysis among the 218 CBF AML samples analyzed for KIT mutations revealed similar complete remission (CR) rates after induction I for KIT + vs. KIT- patients (83% vs. 82%, p=0.796). Five-year event-free survival (EFS) from study entry for KIT + vs. KIT- was 54% and 70%, respectively (p=0.029) with a corresponding overall survival (OS) of 76% vs. 83% (p=0.380). Notably, KIT + patients who achieved CR had a relapse risk (RR) of 45% vs. 23% for KIT- patients (p=0.010). Disease-free survival (DFS) for KIT + vs. KIT- was 51% and 72%, respectively (p=0.021). We also compared the clinical impact of exon 8 vs. exon 17 mutations. Outcomes of CBF AML patients with exon 8 mutations were similar to CBF AML patients without these mutations (OS 90% vs. 80%, p=0.277, EFS 55% vs. 68%, p=0.224, DFS 58% vs. 68%, p= 0.419, RR 42% vs. 26%, p= 0.112). In contrast, outcomes of patients with exon 17 mutations were inferior to those CBF AML patients without exon 17 mutations [OS 64% vs. 84%, p=0.035; DFS 43% vs. 70%, p=0.016) and higher RR was observed (48% vs. 26%, p=0.057). The impact of GO treatment on outcome was subsequently evaluated. KIT + CBF AML patients who did not receive GO had inferior OS and EFS from study entry compared to KIT-patients (OS 64% vs. 86%, p= 0.034, EFS: 46% vs. 69%, p=0.037). Higher RR (55% vs. 31%, p= 0.046) and inferior DFS (45% vs. 66%, p= 0.094) were also observed. In contrast, KIT + and KIT-patients receiving GO treatment had comparable outcomes (OS 88% vs. 80%, p=0.393; EFS 62% vs. 72%, p=0.438) as well as RR (33% vs. 15%, p=0.103) and DFS (57% vs. 77%, p=0.109). Analysis by mutation subtype revealed that outcomes of patients with exon 8 mutations were similar to exon 8 wild-type (WT) patients when treatment did not include GO (OS 81% vs. 80%, p=0.910; EFS 50% vs. 65%, p= 0.185). DFS and RR were also similar (DFS 57% vs. 62%, p= 0.752, RR 43% vs. 36%, p= 0.632). Treatment of exon 8 mutations with GO resulted in significant improvement in OS at 5 years from study entry compared to those without exon 8 mutations (100% vs. 80%, landmark p value 〈 0.001) but other outcome parameters were not significantly improved (EFS 62% vs. 71%, p= 0.707; DFS 58% vs. 75%, p=0.382; RR 42% vs. 16%, p=0.056). For patients with exon 17 mutations, treatment without GO resulted in inferior outcomes when compared to CBF AML patients without exon 17 mutations (OS 56% vs. 85%, p= 0.019; EFS 44% vs. 66%, p=0.154; DFS 33% vs. 65%, p=0.049; RR 67% vs. 32%, p=0.031). Adding GO abrogated this negative impact. Specifically, OS, EFS, DFS and RR for patients with exon 17 mutations were comparable to that of CBF AML patients with WT exon 17 when treated with GO (OS 77% vs. 83%, p= 0.542; EFS 62% vs. 71%, p=0.516; DFS: 56% vs. 74%, p=0.195; RR 22% vs.19%, p=0.898). This analysis suggests that pediatric KIT + CBF AML has negative prognostic impact within the context of AAML0531. This effect was abrogated, particularly for patients with exon 17 mutations, with GO treatment. CD33-targeted agents may be beneficial, at least for a subset of these patients, in future clinical trials. Disclosures Aplenc: Sigma Tau: Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.800.800
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
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  • 9
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    The WT1 synonymous SNP rs16754 Is Associated with Higher mRNA Expression and Predicts Significantly Improved Outcome In Favorable-Risk Pediatric AML: a Report From the Children's Oncology Group
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 950-950
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 950-950
    Abstract: Abstract 950 SNP rs16754, a synonymous single nucleotide polymorphism of the WT1 gene, has recently been reported to have prognostic implications in adult normal-karyotype acute myeloid leukemia (AML). We sought to determine the prevalence and clinical implications of WT1 SNP rs16754 in a large cohort of unselected pediatric AML patients. Available diagnostic bone marrow specimens (N=790) from patients treated on 3 consecutive CCG / COG trials (CCG-2941, CCG-2961, and COG AAML03P1) were analyzed for the presence of SNP rs16754 via direct sequencing of exon 7 of the WT1 gene. SNP status was correlated with disease characteristics, WT1 quantitative mRNA expression level, and clinical outcome. At least 1 copy of the minor SNP allele was detected in 229 (29.0%) patients, 38 (16.6%) of whom were homozygous for the SNP. SNP rs16754 was significantly more common in Asian and Hispanic patients than in Caucasian patients (P 〈 0.001). The SNP was also less common in patients with inv(16) (P=0.043) and more common in patients with -5/del(5q) (P=0.047). Although FLT3/ITD, NPMc, and CEBPA mutations occurred at comparable frequencies in patients with or without the WT1 SNP, WT1 mutations were significantly less common in SNP-positive patients (3.2% vs. 10.4%, P=0.002). WT1 expression levels were evaluated by quantitative RT-PCR in 114 unselected patient samples with known WT1 SNP and mutation status (SNP-positive, n=31; WT1-mutation positive, n=13; one patient harbored both the SNP and a WT1 mutation). Median WT1 expression in WT1 wild-type patients (without SNP rs16754 and negative for a WT1 mutation, n=71) was 40.61 times that of normal marrow controls (range 0.00–2949.42). Median WT1 expression levels were significantly higher in both patients with SNP rs16754 (215.76 fold normal marrow, range 0.00–1399.23, P=0.0214) and in patients with a WT1 mutation (327.14 fold normal marrow, range 54.17–902.3, P=0.0005). Five-year overall survival (OS) for patients with or without the SNP was 60% ± 7% and 50% ± 5%, respectively (P=0.031). In multivariate analysis, the presence of the SNP was an independent predictor of improved OS (HR 0.64, 95% CI 0.47–0.86, P=0.004). Relapse rates were comparable between the 2 groups, but OS after relapse was significantly higher in SNP-positive patients (31% ± 11% vs. 24% ± 6% at 5 years, P=0.034) suggesting a higher rate of salvage success. The prognostic impact was most pronounced in patients with low-risk disease (those with CBF-AML, CEBPA mutation, or NPMc mutation), where five-year OS for those with or without the SNP was 90% ± 8% vs. 64% ± 9% (P=0.001) with a corresponding disease-free survival of 72% ± 13% vs. 53% ± 10% (P=0.041). The high prevalence of WT1 SNP rs16754, and its association with improved outcome, identifies WT1 SNP rs16754 as a potentially important molecular marker of prognosis in pediatric AML. WT1 SNP status, WT1 mutation status, and WT1 expression levels should be evaluated together prospectively in future pediatric AML clinical trials. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.950.950
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
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  • 10
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    Expression Differences of Prognostic Biomarkers in Mononuclear Cells (MNCs) and Malignant Blasts of Acute Myeloid Leukemia (AML) Patients
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 1468-1468
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 1468-1468
    Abstract: Abstract 1468 INTRODUCTION. Many molecular biomarkers have proven to be useful for guiding therapy for AML patients, yet remain inadequate for accurately predicting clinical outcomes. Possible explanations may be that certain biomarkers are more informative for restricted subpopulations of AML patients, or that the results of biomarkers must be interpreted in combination. Another explanation may be that there are methodological limitations in the assays. Most AML biomarkers are currently examined in MNCs, which include non-leukemic cells (lymphocytes, monocytes, and erythroblasts) and leukemic blasts. In addition, cells within the AML blast population display a wide spectrum of differentiation stages. To evaluate the potential impact of the non-leukemic cells in AML samples on the expression of prognostic transcriptional biomarkers, we examined enriched populations of cells from diagnostic AML samples. METHODS. Cryopreserved bone marrow (BM) and peripheral blood (PB) MNCs were obtained from 12 AML patients from the Leukemia Repository at the FHCRC. The cells were thawed and an aliquot was saved for future studies. Remaining cells were sorted for viable AML blasts using a combination of fluorochrome-conjugated antibodies (CD45, CD34, CD38, HLA-DR, and/or CD117) and DAPI. Additional sorts further differentiated the AML blasts into more or less mature (N=4 patients). RNA was extracted using Qiagen AllPrep RNA/DNA kit and reverse transcribed using AMV RT (Invitrogen). Expression of 22 potential prognostic biomarkers was evaluated using commercially available assays (Applied Biosystems) on ABI HT 7900 Fast Real-time PCR system. Fold differences in expression were computed by the comparative CT method, using GUSB to correct for RNA integrity and a pool of RNA from 10 PB from normal donors as a calibrator. RESULTS. Analyses showed marked differences between expression in the unsorted MNCs and AML blasts. Transcript expression was generally the same or more pronounced in AML blasts than in MNCs (e.g., BAALC, Figure 1A). In addition, any transcript differences identified in two populations generally diminished and were less dramatic in AML samples with high blast percentages. However, this was not the case for all samples and/or biomarkers. For example, CEBPA showed similar expression levels in MNCs and AML blasts. Attempts to adjust the signal in MNCs by blast percentages typically did not yield similar transcriptional levels for many samples. Similarly, the expression of examined prognostic biomarkers in subpopulations of AML blasts (mature vs. immature) showed a degree of heterogeneity among the patients. For some biomarkers, the immature population displayed a more pronounced fold difference in expression (e.g. BAALC, Figure1B), while for others, the expression was very similar in examined populations of most patients (e.g. CCNA1). CONCLUSION. As expected, the potential prognostic biomarkers demonstrated variable differences in expression in MNCs and enriched AML blasts, and these differences were even found to occur between the more and less differentiated AML blasts for the same patient. These differences may be of prognostic and predictive significance and may potentially impact the predictive ability of the biomarker. We are expanding these studies to include additional samples and determine if enriching for AML blasts, or even subpopulations within the blasts, may provide a more accurate assessment of prognosis. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.1468.1468
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
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