Characterization of Clonal Plasma Cells in Bone Marrow Biopsy Associated With Newly Diagnosed Acute Myeloid Leukemia: A Pilot Study

PURPOSE Childrens Cancer Group (CCG) protocol 2861 was designed to test the feasibility of aggressively timed induction therapy followed by autologous or allogeneic bone marrow transplantation (BMT) as the sole postremission therapy for newly diagnosed children with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). PATIENTS AND METHODS Between April 1988 and October 1989, 142 patients were eligible for study. All patients entered received a timing-intensive five-drug induction of dexamethasone, cytarabine (Ara-C), thioguanine, etoposide, and daunorubicin (DCTER) over 4 days with a second cycle administered after 6 days of rest, irrespective of hematologic status at that time. Most patients subsequently received a second two-cycle induction course. Those who achieved remission were eligible for bone marrow ablative therapy with busulfan and cyclophosphamide, followed by 4-hydroperoxy-cyclophosphamide (4-HC)-purged autologous or allogeneic BMT rescue. RESULTS One hundred eight (76%) patients achieved remission: 19 (13%) died of complications of the leukemia and/or chemotherapy, and 15 (11%) failed to achieve remission. Seventy-four patients subsequently underwent BMT with either autologous (n = 58) or allogeneic (n = 16) rescue. For patients who received autologous rescue with 4-HC-purged grafts, the actuarial disease-free survival (DFS) rate at 3 years from the day of transplant is 51%, compared with 55% for patients who received allogeneic grafts (P = .92). At 3 years, the overall actuarial survival rate for all 142 patients entered on this study is 45%, with an event-free survival (EFS) rate of 37%. Adverse prognostic factors for outcome included an elevated WBC count or the presence of CNS leukemia at the time of AML diagnosis. CONCLUSION Results suggest that aggressively timed induction therapy followed by marrow ablation and BMT rescue with either autologous or allogeneic grafts for children with newly diagnosed AML or MDS is both feasible and effective.

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Clinical Activity, Pharmacokinetics, and Pharmacodynamics of Sorafenib In Pediatric Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v116.21.1073.1073 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1073-1073

Author(s):

Hiroto Inaba ◽

Jeffrey E Rubnitz ◽

Elaine Coustan-Smith ◽

Lie Li ◽

Brian D Furmanski ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Leukemic Cells ◽

Clinical Activity ◽

Newly Diagnosed ◽

Pediatric Acute Myeloid Leukemia ◽

Rtk Signaling ◽

Acute Myeloid

Abstract Abstract 1073 Background: Aberrant receptor tyrosine kinase (RTK) signaling arising from genetic abnormalities, such as FLT3-internal tandem duplications (FLT3-ITD), is an important mechanism in the development and growth of acute myeloid leukemia (AML) and is often associated with a poor outcome. Hence, inhibition of RTK signaling is an attractive novel treatment option, particularly for disease that is resistant to conventional chemotherapy. We evaluated the clinical activity of the multikinase inhibitor sorafenib in children with de novo FLT3-ITD–positive AML or relapsed/refractory AML. Methods: Fourteen patients were treated. Six patients with newly diagnosed FLT3- ITD–positive AML (aged 9–16 years; median, 12 years) received 2 cycles of remission induction therapy and then started sorafenib (200 mg/m2 twice daily for 20 days) the day after completing induction II (low-dose cytarabine, daunorubicin, and etoposide). Nine patients (aged 6–17 years; median, 9 years) with relapsed AML (including one treated on the above regimen) received sorafenib alone (2 dose levels; 200 and 150 mg/m2) twice daily for the first week of therapy, concurrently with clofarabine and cytarabine on days 8–12, and then alone from days 13 to 28. Sorafenib pharmacokinetics were analyzed at steady-state on day 8 of sorafenib in patients with newly diagnosed AML and on day 7 in patients with relapsed AML. In patients with relapsed AML, the effect of sorafenib on signaling pathways in AML cells was assessed by flow cytometry. Results: All 6 newly diagnosed patients, including 2 whose AML was refractory to induction I, achieved a complete remission (CR) after induction II; 5 had negative minimal residual disease (MRD; <0.1% AML cells in bone marrow) after induction II. Both patients in this group who relapsed achieved second remissions, one with sorafenib alone and one on the relapse regimen described above. Of the 9 patients with relapsed AML, 6 (4 with FLT3-ITD) were treated with sorafenib 200 mg/m2. All 6 had a >50% decrease in blast percentage and/or bone marrow cellularity after 1 week of sorafenib. After concurrent sorafenib and chemotherapy, 5 of the 9 patients with relapsed AML achieved CR (2 had negative MRD) and 2 achieved a partial remission (PR; 5%-25% AML cells in bone marrow); all 4 patients with FLT3-ITD had a CR or PR. After sorafenib treatment, 6 patients underwent HSCT while 2 with FLT3-ITD who could not receive HSCT were treated with single-agent sorafenib and have maintained CR for up to 8 months. Hand-foot skin reaction (HFSR) or rash occurred in all patients and improved with cessation of sorafenib. Dose-limiting toxicity (DLT, grade 3 HFSR and/or rash) was observed in 3 of the 6 patients with relapsed AML treated with 200 mg/m2 of sorafenib; no DLT was observed at 150 mg/m2. The effect of sorafenib on downstream RTK signaling was tested in the leukemic cells of 4 patients: in most samples, phosphorylation of S6 ribosomal protein and 4E-BP1 was inhibited. The mean (± SD) steady-state concentration (Css) of sorafenib was 3.3 ± 1.2 mg/L in the newly diagnosed group and 6.5 ± 3.6 mg/L (200 mg/m2) and 7.3 ± 3.6 mg/L (150 mg/m2) in those with relapsed AML. In both groups, the mean conversion of sorafenib to sorafenib N-oxide was 27%-35% (approximately 3 times greater than previously reported), and mean sorafenib N-oxide Css was 1.0–3.2 mg/L (2.1-6.7 μM). In a 442-kinase screen, the inhibitory profiles of sorafenib N-oxide and sorafenib were similar, and FLT3-ITD phosphorylation was potently inhibited by both forms (sorafenib N-oxide Kd = 0.070 μM; sorafenib Kd = 0.094 μM). Sorafenib N-oxide inhibited the growth of an AML cell line with FLT3-ITD (IC50 = 0.026 μM) and 4 AML cell lines with wild-type FLT3 (IC50 = 3.9–13.3 μM) at approximately half the potency of sorafenib. Conclusion: In children with de novo FLT3-ITD and relapsed/refractory AML, sorafenib given alone or with chemotherapy induced dramatic responses and inhibited aberrant RTK signaling in leukemic cells. Sorafenib and its active metabolite (sorafenib N-oxide) likely contribute to both efficacy and toxicity. These results warrant the incorporation of sorafenib into future pediatric AML trials. Disclosures: Inaba: Bayer/Onyx: Research Funding. Off Label Use: Sorafenib and clofarabine: both used for treatment of pediatric acute myeloid leukemia.

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Plasma Exosomes As Markers of Residual Disease in Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.2357.2357 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2357-2357

Author(s):

Michael Boyiadzis ◽

Chang Sook Hong ◽

Theresa L Whiteside

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Complete Remission ◽

Protein Content ◽

Myeloid Leukemia ◽

Residual Disease ◽

Molecular Profile ◽

Consolidation Therapy ◽

Newly Diagnosed ◽

Acute Myeloid

Abstract Background: Exosomes are virus-size (30–100 nm in diameter) membrane-bound microvesicles that are formed within the endocytic compartments and via fusion of multivesicles bodies are released into extracellular space. The exosomal cargo includes proteins/glycoproteins expressed on the cell membrane as well as molecules and soluble factors present in the cytosol of parental cells. While exosome secretion occurs under physiologic conditions, and all cells are capable of their release, tumor cells are avid exosome producers. Patients newly diagnosed with acute myeloid leukemia (AML) prior to any therapy have higher levels of exosomes compared to normal controls (NC). We hypothesize that the molecular content of isolated exosomes, which are thought to mimic that of leukemic blasts, could be informative about the presence in the bone marrow of leukemic blasts that might avoid detection by conventional hematopathological assays. Methods: Samples of venous blood (20-50 mL) were obtained from patients newly diagnosed with AML prior to any treatment (n=13), after completion of initial induction chemotherapy in patients who achieved complete remission (n=8), during consolidation therapy and age-matched healthy volunteers. Exosome fractions were isolated from plasma by exclusion chromatography on Sephadex G50 columns followed by ultracentrifugation. Exosome protein content was determined and expressed in µg protein/mL plasma. Isolated exosomes were characterized by western blots for expression of classical exosomal markers and for expression of novel myeloid cell surface markers associated with AML, interleukin-3 receptor a chain (CD123) and C-type lectin-like molecule-1 (CLL-1). Results: The exosome fractions isolated from AML patients’ plasma at diagnosis had a considerably greater mean protein content (81.5 ± 10.8 μg protein/mL plasma) than did exosome fractions isolated from the plasma of NC (13.1 ± 2.4 μg protein/mL plasma) with p < 0.005. The molecular profile of exosomes isolated from plasma of AML patients at diagnosis was distinct from that of exosomes isolated from plasma of NC. In addition to classical exosomal markers (MHC class I molecules, LAMP-1, CD81) exosomes isolated from AML patients at diagnosis contained CD34, CD117, CD123 and CLL-1. The exosome fractions isolated from the patients’ plasma who achieved complete remission (n=8) remained elevated, similar to the levels at the time of AML diagnosis (78.5 vs 77.5 μg protein/mL plasma). Exosomes in AML patients who achieved complete remission and in patients receiving consolidation therapy when leukemic blasts are undetectable in the bone marrow by conventional hematopathological methods contained CD123 and CLL-1. Conclusions: Exosomes in AML patients have a unique and distinctive molecular profile. The exosomal profile suggest the presence of residual disease in patients considered to have achieved complete remission by conventional hematopathologic assays. Disclosures No relevant conflicts of interest to declare.

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Mutation Analysis in 276 Cases of Newly Diagnosed Acute Myeloid Leukemia By Next Generation Sequencing: Association with Established Prognostic Variables and MRC Risk Groups

Blood ◽

10.1182/blood.v126.23.1392.1392 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1392-1392

Author(s):

Parsa Hodjat ◽

Kankana Ghosh ◽

Priyanka Priyanka ◽

Beenu Thakral ◽

Keyur P. Patel ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Next Generation Sequencing ◽

Mutation Analysis ◽

Myeloid Leukemia ◽

Risk Groups ◽

Newly Diagnosed ◽

Generation Sequencing ◽

Acute Myeloid ◽

Who 2008

Abstract INTRODUCTION Acute myeloid leukemia (AML) is known to have numerous genomic aberrations that predict response to treatment and overall survival. We aimed to assess various mutations in newly diagnosed AML cases by next generation sequencing (NGS) and their association with various well-established clinicopathologic parameters and Medical Research Council (MRC) risk groups. MATERIALS AND METHODS We performed molecular studies on DNA extracted from bone marrow aspirate specimens in 276 newly diagnosed treatment na•ve AML patients presenting at a single referral institution from 08/2013 to 03/2015 as part of routine clinical work up in a CLIA certified molecular diagnostics laboratory. Cases met criteria for AML per WHO 2008 criteria. The entire coding sequences of 28 genes (ABL1, ASXL1, BRAF, DNMT3A, EGFR, EZH2, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, IKZF2, JAK2, KIT, KRAS, MDM2, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1) were sequenced using a NGS-based custom-designed assay using TruSeq chemistry on Illumina MiSeq platform. FLT3 internal tandem duplications (ITD) and codon 835/836 point mutation were detected by PCR followed by capillary electrophoresis. CEBPA mutation analysis was performed on 262 patients by PCR followed by Sanger sequencing. Cases were categorized as favorable, intermediate and adverse groups as per revised MRC cytogenetic risk group classification. RESULTS Median age was 67 years. Patients included 167 (60.5%) males and 109 (39.5%) females. 38 (14%) and 6 (2%) patients had prior diagnosis of myelodysplastic syndrome and myeloproliferative neoplasms respectively. Hematologic parameters are as follows [median (range)]: Hb 8.7 g/dL (2.8-13.9), platelets 50.5 K/μ L (1-1109), WBC 5.4 K/μ L (0.4-620.4), ANC 0.9 K/μ L (0-145.7), AMC 0.3 K/μ L (0-98.1). Bone marrow (BM) blast % [median (range)] was 45.5% (5-96). LDH was 733 IU/dL (225-13156). Of 275 patients with cytogenetic analysis performed, 98 (35.64%) had diploid karyotype, 75 (27.27%) had one, 38 (13.82%) had two, 8 (2.91%) had three, 56 (20.36%) had > three abnormalities, 75 (27.27%) had monosomies and 62 (22.55%) had trisomies. Of 34 cases classified as AML with recurrent cytogenetic abnormalities per WHO 2008, 10 (3.64%) had t(8;21), 13 (4.73%) had inv(16), 1 (0.36%) had t(15;17), 3 (1.09%) had inv (3), 4 (1.45%) had t(9;11)(p22;q23) and 3 (1.09%) had t(6;9)(p23;q34). MRC risk categorization of the cases was as follows: favorable 24 (8.72%), intermediate 161 (58.55%) and adverse 90 (32.73%). Mutations identified by NGS are as detailed in Table 1. Of 56 patients with FLT3 mutations detected by PCR, the breakdown is as follows: FLT3 ITD (39, 14.13%), FLT3 D835 (16, 5.80%), FLT3, ITD + D835 (1, 0.36%). Of 262 patients assessed, CEBPA mutation was detected in 26 (9.92%). Thirty one (11.23%) cases had no mutations detected in the genes analyzed by NGS or PCR, 93 (33.70%) had mutations in one, 80 (28.98%) in two, 42 (15.22%) in three and 30 (10.87%) in > three genes. We found positive associations between mutated genes and various parameters as detailed in Table 2. CONCLUSIONS: AML is a heterogeneous group of myeloid neoplasms at the genetic level. Multiple genetic mutations in a large subset of cases likely indicate clonal evolution. A subset of mutations has significant association with well-established clinico-pathologic parameters like WBC. With longer follow-up, we could use this data to refine prognostic models for AML. Table 1. Genes Number of Cases Percentage of Cases FLT3 61 22.10 NPM1 48 17.39 NRAS 48 17.39 DNMT3A 47 17.03 TP53 45 16.30 IDH2 40 14.49 IDH1 33 11.96 TET2 32 11.59 ASXL1 30 10.87 RUNX1 30 10.87 PTPN11 13 4.71 KRAS 11 3.99 KIT 8 2.90 WT1 8 2.90 GATA2 7 2.54 EZH2 6 2.17 JAK2 4 1.45 MPL 2 0.72 ABL1 1 0.36 EGFR 1 0.36 GATA1 1 0.36 IKZF2 1 0.36 MDM2 1 0.36 MLL 1 0.36 MYD88 1 0.36 NOTCH1 1 0.36 Table 1. Mutated genes p value Hb NRAS, NPM1 <0.05, <0.04 Platelets TP53, IDH2 <0.03, <0.02 WBC FLT3, NRAS, TP53 <0.05, <0.05, <0.05 AMC NRAS, NPM1, TP53 <0.001, <0.02, <0.02 ABC FLT3 NPM1 <0.049, <0.02 PB blast % FLT3, NPM1, TP53, CEBPA <0.000, <0.002, <0.005, <0.000 BM blast % FLT3, NRAS, NPM1, TP53, IDH1, CEBPA >0.000, <0.0000, <0.014, <0.004, <0.002, <0.012 AMC: absolute monocyte count, ABC: absolute basophil count, PB: peripheral blood, BM: bone marrow Disclosures No relevant conflicts of interest to declare.

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Comparison of blast clearance in peripheral blood and day 14 bone marrow biopsy for evaluation of disease response after 7+3 induction in acute myeloid leukemia (AML).

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.15_suppl.7052 ◽

2018 ◽

Vol 36 (15_suppl) ◽

pp. 7052-7052

Author(s):

Fahrettin Covut ◽

Divya Gupta ◽

Raisa Pinto ◽

Nina Dambrosio ◽

Najla H. El Jurdi ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Bone Marrow Biopsy ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Disease Response ◽

Acute Myeloid

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Will deeper characterization of the landscape of immune checkpoint molecules in acute myeloid leukemia bone marrow lead to improved therapeutic targeting?

Cancer ◽

10.1002/cncr.32042 ◽

2019 ◽

Vol 125 (9) ◽

pp. 1410-1413 ◽

Cited By ~ 1

Author(s):

Esten N. Vandsemb ◽

Tae Kon Kim ◽

Amer M. Zeidan

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Immune Checkpoint ◽

Myeloid Leukemia ◽

Therapeutic Targeting ◽

Immune Checkpoint Molecules ◽

Acute Myeloid

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Imatinib Mesylate-Induced Complete Cytogenetic Response in Acute Myeloid Leukemia with t(5;12)(q33;p13).

Blood ◽

10.1182/blood.v108.11.4414.4414 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4414-4414

Author(s):

William Blum ◽

Guido Marcucci ◽

Hollie Devine ◽

Rebecca Klisovic

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Imatinib Mesylate ◽

Bone Marrow Biopsy ◽

Myeloid Leukemia ◽

Cytogenetic Response ◽

Cytotoxic Chemotherapy ◽

Pleural Effusions ◽

Complete Cytogenetic Response ◽

Acute Myeloid

Abstract Imatinib mesylate (IM) is a tyrosine kinase inhibitor (TKI) with established activity in chronic myelogenous leukemia (CML) due to targeted disruption of constitutively activated bcr-abl TK in the leukemic clone. IM also inhibits several other TKs that are aberrantly activated in other diseases such as c-kit in gastrointestinal stromal cell tumor, platelet-derived growth factor receptor-α (PDGFR-α) in hypereosinophilic syndrome, and PDGFR-β in chronic myelomonocytic leukemia (CMML). In CMML, PDGFR-β at 5q33 is typically fused with the ETV6 locus (formerly TEL) at 12p13 as t(5;12)(q33;p13). We report a case of IM-induced complete cytogenetic response in a patient with acute myeloid leukemia (AML) who presented with multiple extramedullary sites of disease and a complex karyotype of 49,XX,t(5;12)(q33;p13),+10,+11,+19. On transfer to our facility shortly after initial presentation, the patient had fatigue and cervical lymphadenopathy with a white blood cell count of 6,200/uL (43% segs, 36% lymphs, 9% monos, 11% eos, rare blasts) and was platelet transfusion dependent. She had no known prior history of CMML or myeloproliferative disorder, though she had increasing fatigue for 6 months. Excisional biopsy of a cervical node demonstrated a myeloid sarcoma; bone marrow biopsy confirmed AML, subtype M4 by FAB classification, with 31% blasts. There was mild eosinophilia in the marrow. The karyotype from the node was the same as in the marrow. CT scans demonstrated extensive lymphadenopathy (including neck, axilla, mediastinum, abdomen), splenomegaly measuring 15cm, and bilateral pleural effusions. After induction chemotherapy consisting of cytarabine, daunorubicin, etoposide, and tipifarnib (refused further tipifarnib after receiving only one dose) on an NCI-sponsored clinical trial, the patient had morphologic remission in the bone marrow but persistent t(5;12) in 20/20 cells as well as persistent lymphadenopathy, splenomegaly, and pleural effusions. The other cytogenetic abnormalities had resolved. Given prior reports of IM-responsive CMML in patients with t(5;12)(q33;p13), the patient began IM at 400mg daily. After one month of IM, repeat bone marrow biopsy demonstrated morphologic CR and only 6/20 cells with t(5;12). On the basis of this improvement, a clinical decision was made to postpone further cytotoxic chemotherapy-based consolidation and continue IM while an allogeneic stem cell donor could be identified. After 10 weeks of IM, bone marrow biopsy demonstrated continued CR with normal cytogenetics. Flouresence-in-situ-hybridization (FISH) with an ETV6 breakapart probe was also negative (probe was positive on the diagnostic sample in 66% of cells). The patient had complete resolution of the pleural effusions with no palpable adenopathy or splenomegaly and a performance status of 100%. Allogeneic transplantation in first remission was recommended due to the presence of multiple additional cytogenetic changes/ extramedullary disease at diagnosis and the availability of a 10/10 HLA allele matched donor. Molecular characterization of PDGFR-β transcript levels in this patient is ongoing. This case suggests that efficacy of IM in t(5;12) positive AML may be analogous to its efficacy in t(9;22) positive blast phase CML. Targeted inhibition of aberrantly activated PDGFR-β with IM may be effective in clearing residual disease in the marrow following cytotoxic chemotherapy for AML patients with t(5;12)(q33;p13). IM may also be a useful adjunct to cytotoxic treatment in AML patients with aberrant activation of other IM-sensitive TKs.

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