Microgranular Variant Of Acute Promyelocytic Leukemia Research Articles

Specific Patterns of Molecular Mutations Determine the Morphologic Differentiation Stages in Acute Myeloid Leukemia (AML)

Background: The FAB classification of AML was based on the differentiation state and the involved cell lineages and recognized some close associations between genetic abnormalities and cytomorphology: CBFB-MYH11 and AML with abnormal eosinophils, PML-RARA and acute promyelocytic leukemia; RUNX1-RUNX1T1 and AML with maturation (FAB subtype M2). Associations with molecular mutations have not been examined in detail yet.Aim: Evaluate the impact of molecular mutations on the AML differentiation state.Material and Methods: The cohort comprized 3,703 patients with de novo AML (median age 67 yrs; range: 18.1-100.5 yrs). All cases were analyzed by cytomorphology and molecular mutation screening for 12 genes by next generation or Sanger sequencing in our laboratory. Based on cytomorphology and immunophenotyping we categorized the cohort into 11 subgroups: 1. AML with minimal differentiation (according to the FAB classification: M0; n=192), 2. AML without maturation (M1; n=1,015), 3. AML with maturation (M2; n=1,324), 4. acute promyelocytic leukemia (APL) with PML-RARA (M3; n=124), 5. the microgranular variant of APL (M3v, n=110), 6. acute myelomonocytic leukemia (M4; n=604), 7. acute myelomonocytic leukemia with abnormal eosinophils and CBFB-MYH11 (M4eo; n=81), 8. acute monoblastic leukemia (M5a; n=86), 9. acute monocytic leukemia (M5b; n=63), and 10. acute erythroid leukemia (M6; n=101). Cases with acute megakaryoblastic leukemia (M7; n=3) were not considered for statistical evaluation. All 3,703 cases were analyzed for FLT3-ITD and FLT3-TKD. All cases except M3/M3v and M4eo (n=3,487) were screened for NPM1 und MLL-PTD. Data on the mutation status of the following genes was available: FLT3-ITD (n=3,703), FLT3-TKD (n=3,703), CEBPA (n=2,684), RUNX1 (n=2,320), WT1 (n=2,224), ASXL1 (n=1,591), DNMT3A (n=1,294), NRAS (n=1,232), TP53 (n=1,046) and TET2 (n=974). Statistical significance of associations between the mutation frequencies and AML subtypes were assessed by Fisher’s exact test and corrected according to Benjamini-Hochberg (1995) to compensate for multiple testing.Results: The mutation rates of the analyzed genes are in line with published data.Total cohort: Significant associations were observed between the mutation frequencies of distinct genes and the differentiation stage of the AML. Figure 1 depicts genes showing significantly different mutation rates within distinct AML subtypes compared to the rate of the total cohort. In detail, mutations in RUNX1 were most frequently observed in M0 (43.6%) and M2 (24.0%) and were nearly absent in M5a (3.5%) and M5b (0%). The highest TP53 mutation rates were observed in M6 (35.3%) and M2 (15.0%), the lowest in M4 (3.0%) and M1 (5.7%). NPM1 mutations were significantly more frequent in M5b (69.8%), M4 (56.3%), M5a (47.7%) and M1 (42.1%) than in M2 (24.2%), M6 (20.8%), M0 (3.1%), and absent in M3, M3v, and M4eo, respectively.NPM1 mutated subcohort (n=1,200): The overall DNMT3A mutation rate in this subcohort was 49.1%. It was significantly higher in M5b (75.0%) and M4 (61.6%) and significantly lower in M1 (40.6%), M5a (22.2%), and M6 (13.3%). Contrary, RUNX1 (overall frequency in NPM1mut cases 1.2%) and ASXL1 (overall 2.5%) mutations were more frequent in M5a (8.3% and 34.8%, respectively), while FLT3-ITD was more frequent in M1 (52.5%), and WT1 mutations more frequent in M6 (26.7%).CEBPA double mutated subcohort (n=108): ASXL1 and RUNX1 mutations were 5- and 7-fold more frequent in M2 than in M1, but did not reach significance.Conclusions:Specific patterns of molecular mutations are linked to distinct AML differentiation stages: 1) RUNX1 and ASXL1 with M0 2) NPM1, ASXL1 and TET2 with M5a, and 3) NPM1 and DNMT3A with M5b. 4) The differentiation stage of NPM1 mutated AML is driven by the presence of additional mutations: DNMT3A mutations lead to a (myelo-)monocytic differentiation, while mutations in RUNX1 or ASXL1 lead to a monoblastic phenotype. 5) FLT3-ITD is most frequent in NPM1 mutated AML without maturation and WT1 mutations are most frequent in acute erythroid leukemia bearing NPM1mut. [Display omitted] DisclosuresRose:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o.: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Clinical Characteristics and Outcomes In Patients With Acute Promyelocytic Leukemia (APL) and Hyperleukocytosis

BackgroundThere is limited information on the best management of hyperleucocytosis at presentation in patients with acute promyelocytic leukemia (APL) and few studies have evaluated the clinical features and treatment options in this subset of patients (pts). MethodsWe reviewed the medical records of 233 pts with a diagnosis of APL at University of Texas - MD Anderson Cancer Center between January 1990 and December 2011. Twenty-nine (12%) had a white cell count (WBC) ≥ 50 x 109/L at presentation. Kaplan-Meier methods and log-rank tests were used to assess survival. ResultsAmong the 29 pts, 13 (45%) were male and median age at presentation was 48 years (29 - 77). Thirteen pts (45%) had the microgranular variant of APL. Karyotype analysis was available in 23 pts (79.3%). All 23 pts harbored the t(15:17)(q22; q21). Additional cytogenetic abnormalities involved chromosome 9 in 3 (10%), chromosome 21 in 2 (7%) and others in 6 (18%) each. Median PML-RARA transcript value was 24.0 (0.13 - 42.7). FLT3 analysis was performed in 11 pts presenting after 2003 with 100% having FLT3-internal tandem duplication and one having both FLT3-ITD and FLT3-tyrosine kinase domain mutations. No RAS or c-KIT mutations identified. Median WBC count, peripheral promyelocyte percentage, hemoglobin and platelet count at presentation were 85.5 x 109/L (53.8 - 194.8), 86.5% (0 - 97), 9.60 g/dL (7.7 - 12.8), and 25 x 109/L (10 - 124), respectively. Median bone marrow (BM) cellularity, BM promyelocyte, and BM blasts at presentation were 95% (75 - 100), 82% (0 - 90), and 5% (0 - 97), respectively. 23 (79%) pts had clinical evidence of leucostasis at presentation including hypoxia in 10 (34%), hemoptysis or diffuse alveolar hemorrhage in 6 (21%), stroke or intracranial bleeding in 4 (14%), intraocular bleeding in 4 (14%), and gastrointestinal bleeding in 4 (14%). Chemical DIC seen in 27 pts (93%) with median fibrinogen of 243 mg/dL (125 - 662), median PT of 16.6 seconds (12.6 - 24.2), median PTT of 25 seconds (21.0 - 33.8) and median INR of 2.21 (1.56 - 2.95). Treatment regimens included all-trans retinoic acid (ATRA)/arsenic trioxide (ATO) combinations in 15 (52%) patients including ATRA+ ATO in 4 (14%) and ATRA+ATO+GO (gemtuzumab ozogamicin) in 11 (38%). Non-ATRA/ATO regimens were used in 14 (48%) patients including ATRA+idarubicin (IDA) in 8 (27%), ATRA+GO in 4 (14%), and ATRA+IDA+GO in 2 (7%), respectively. Leukapheresis was performed in 11 pts (38%) with a median of 3 leukapheresis procedures (1 - 6). Median WBC at initiation and completion of leukapheresis were 106.0 (80.2 - 194.8) and 26.1 (1.7 - 35.9), respectively. 6 pts died within 4-weeks of initiating therapy. All remaining 23 pts achieved morphological complete remission (CR) after induction with median time to CR of 21.5 days (15 - 35). 6 pts relapsed and 5 achieved a second CR with salvage regimens including allogeneic transplant in 2 pts. Median OS for the entire group is 67 months. CR rate and 3-year overall survival (OS) for the pts who underwent leukapheresis were 82% and 73% versus 78% and 67% for those who did not receive leukapheresis (P=0.79 and P=0.64; respectively). CR rate and 3-year OS for the pts treated with ATRA/ATO combinations were 100% and 100% versus 57% and 35% for those treated with non-ATRA/ATO combinations (P=0.004 and P=0.002, respectively). ConclusionsATRA/ATO based combinations are superior to other therapeutic regimens in patients with APL who have hyperleukocytosis and WBC ≥ 50 x 109/L at presentation with an improved CR rate and improved 3-year OS.Table 13-year OS by patient characteristicsCharacteristicsN3-year survival %P-valueAge</=6524790.12>65520GenderMale13690.94Female1668Cytogeneticst(15;17) alone17880.12t(15;17) + Other650Clinical StasisYes23700.58No667WBC</=10017770.64>1001257Platelets</=2011620.9>201872Fibrinogen</=20011640.43>2001675LeukapheresisYes11730.64No1867RegimenATRA/ATO151000.002Non-ATRA/ATO1435▪ Disclosures:Kantarjian:Sanofi-Aventis: Research Funding. Faderl:Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Garcia-Manero:Novartis Pharmaceutical: Research Funding. Verstovsek:Incyte Corporation: Research Funding. Ravandi:Sunesis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Teva: Consultancy, Honoraria; Pfizer: Honoraria; Merck: Research Funding; Bayer/Onyx: Consultancy, Honoraria; EMD Serono: Research Funding; Medimmune: Research Funding; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria.

All-Trans-Retinoic Acid andBleeding/Thrombosis

Hematology-Oncology Department Ospedali Riuniti Largo Barozzi, 1 24128 Bergamo (Italy) Tel. 39 035 269492, Fax 39 035 266659 E-Mail annafalanga@yahoo.com All-trans-retinoic acid (ATRA) inhibits cell growth and proliferation by inducing cyto-differentiation and/or apoptosis in several cell types. These effects have become a therapeutic objective in human cancers. In addition to these functions, ATRA has been shown to affect cellular hemostatic properties [1]. Much understanding in this field has come from the clinical and experimental studies of human acute promyelocytic leukemia (APL), a distinct subtype of acute myelogenous leukemia (AML-M3), cytogenetically characterized by the balanced reciprocal translocation between chromosomes 15 and 17. In these cells, the fusion of the nuclear retinoic acid receptor (RAR ) gene on chromosome 17 with part of the PML gene on chromosome 15 results in the expression of a chimeric PML/RAR protein, which is involved in the leukemogenesis and is the target for the myeloid differentiation effect induced by ATRA. The disease typically presents with a life-threatening hemorrhagic diathesis, which is worsened by cytotoxic chemotherapy. The bleeding disorder is particularly severe in the microgranular variant of APL (M3v), which is characterized by marked hyperleukocytosis. Before the introduction of ATRA for the management of APL patients, fatal hemorrhages due to the associated coagulopathy were a major cause of induction remission failure. In a large retrospective study, the overall remission rate was 62% and the prevalence of hemorrhagic deaths in induction 14% [2]. The use of ATRA for the remission induction therapy of APL has raised the complete remission rate to greater than 90%, together with a rapid resolution of the coagulopathy, without causing bone marrow hypoplasia. ATRA promotes the terminal differentiation of leukemic promyelocytes, which is accompanied by prompt improvement of the coagulopathy typical of this disease [3]. Consistent with that, normalization in the plasma levels of hypercoagulation markers and downregulation of the two major blast cellassociated procoagulants (i.e. tissue factor, TF, and cancer procoagulant, CP) were observed [4]. A number of laboratory studies have subsequently confirmed the decrease or normalization of clotting and fibrinolytic variables during the first 1 or 2 weeks of therapy with ATRA. Inspite of that, the impact of ATRA therapy on early hemorrhagic deaths and CR rate in APL remains uncertain compared to chemotherapy alone. In nonrandomized studies, APL patients administered ATRA showed 9% to 20% improvement of CR rate and 5% to 6% reduction of early hemorrhagic deaths, compared to historical controls receiving conventional chemotherapy [1, 5–8]. These preliminary findings have been more recently confirmed by randomized clinical trials. APL patients treated with different combinations of ATRA plus chemotherapy show a prevalence of early hemorrhagic deaths from 2.4% to 6.5% [9–13]. However, both nonrandomized and randomized clinical trials clearly show an improvement of event free and overall survival in patients receiving induction therapy All-Trans-Retinoic Acid and Bleeding/Thrombosis

Translocation (15;17) and trisomy 21 in the microgranular variant of acute promyelocytic leukemia

Cytogenetic abnormalities in acute myelogenous leukemia have been identified as one of the most important prognostic factors. Favorable chromosomal changes such as inv(16), t(8;21), and t(15;17) are associated with higher rates of complete remission and event-free survival. Translocation t(15;17) characterizes acute promyelocytic leukemia (APL) (French-American-British [FAB] class M3) in almost all patients. Secondary chromosomal abnormalities are also present in approximately one-third of patients with newly diagnosed APL. We present a 26-year-old Hispanic man diagnosed with the microgranular variant of APL (FAB class M3v) whose initial cytogenetics included t(15;17) and trisomy 21. The prognostic implications of trisomy 21 and other secondary cytogenetic aberrations in APL are reviewed. To our knowledge, this is the first reported case of trisomy 21 with t(15;17) in the microgranular variant of APL.

An immunofluorescent assay for acute promyelocytic leukemia cells.

Sequential treatment with all-trans retinoic acid followed by chemotherapy significantly improves the long-term survival of patients who have acute promyelocytic leukemia (APL). Consequently, a simple and accurate test is needed to establish the diagnosis of APL and to identify those patients having a relapse of the disease. We describe an accurate, 2-hour indirect immunofluorescent assay for identifying APL cells in bone marrow specimens. The assay uses the PML (PG-M3) murine monoclonal antibody that is directed against the amino-terminal portion of the PML gene product. We observed a distinctive, finely speckled pattern of fluorescence in the NB4 cell line (a positive control), as well as in 15 clinical specimens that were confirmed to have APL by cytogenetic, cytochemical, and immunophenotypic studies, including four cases of microgranular variant of APL. By contrast, a coarse globular pattern of fluorescence was observed in 53 other clinical specimens that did not contain APL. When we performed dilution studies using artificial mixtures of APL cells with normal bone marrow cells, we detected as few as 5% APL cells in the mixture. Finally, there was complete concordance between the immunofluorescent assay and a polymerase chain reaction-based assay for the PML-retinoic acid receptor alpha chimeric gene in 12 other clinical specimens. We conclude that the immunofluorescent assay for PML protein is a rapid, sensitive, and accurate method for determining the presence of APL cells in clinical specimens. This assay therefore should be considered as a cost-effective alternative to other diagnostic tests, such as karyotyping or polymerase chain reaction, for the diagnostic evaluation of APL.