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Case 398 Submitting Author: Hutchison, Robert E, MD Institution: SUNY Upstate Medical University Additional authors: Constance K. Stein, Ph.D., Theresa C. Gentile, M.D., Ph.D., Bhuvaneswari Ramkumar, M.D. History • The patient is a 48 year old female who presented to her doctor with symptoms consistent with a sinus infection with head congestion, shortness of breath and cervical lymphadenopathy which did not improve with antibiotics. She was found to have a white blood cell count of 20,000 /ul with peripheral blasts, anemia and thrombocytopenia. Details • RPIC bone marrow aspiration and biopsy. • Aspirate films (push preps) and clot preparation prepared at the bedside • RPIC trephine biopsy and touch imprints made at the bedside. • The biopsy and clot were fixed in B5 fixative for two hours and then transferred to 70% ETOH prior to processing. • Peripheral blood in EDTA for CBC and blood films. • Aspirate films and touch imprints stained manually with Wright Giemsa; blood films with an automated stainer. • Sections stained with H&E. Blood • WBC=20.2 K/ul – 85.5% blasts – 14.5% lymphocytes – 8 NRBC/100 WBC • • • • Hgb=7.5 g/dl RBC=2.24 M/ul MCV=101.8 fl plt=61 K/ul – Blasts showed high N/C ratio, diffuse chromatin with 1-2 nucleoli and occasional Auer rods. Blood film BM aspirate • Hypercellular marrow – 41.4% erythroid precursors with moderate megaloblastic/dysplastic features – 50.8% blasts • Auer rods were frequent and sometimes multiple. – 6.8% lymphocytes – 1.0% plasma cells – Occasional megakaryocytes BM aspirate BM aspirate Erythroid dysplasia Biopsy/clot sections IMMUNOHISTOCHEMISTRY AND FLOW CYTOMETRY • Flow cytometry: – Blasts positive for: • CD38, CD117, CD13, CD33, MPO (98%) • dim/partial CD34 (29%) and CD15 (49%). – negative for: • CD14, CD64, cCD3, cCD79a, cCD22 and Tdt. • Immunohistochemistry: – Cytoplasmic and nuclear NPM NPM Karyotype: 47,XX,+4(9); 46,XX(11) FISH • chr. 4: nuc ish (D4Z1x3)(64/100) • FISH showed 64% of cells with an additional signal for chromosome 4 indicating trisomy 4. MOLECULAR FINDINGS • Positive for NPM1 (exon 12) mutation, 39.88% • PCR amplification performed using NPM intron 11 forward primer and 6-fam-labeled NPM exon 12 reverse primer - Quest • Negative for FLT3 • FLT3 length mutation (including internal tandem repeats) as well as point mutations at D835 and I836 within the FLT3 tyrosine kinase domain– SUNY-UMU INTERESTING FEATURES • AML with trisomy 4 as the sole cytogenetic abnormality is a rare to uncommon disease that has been described but not classified among AML with recurrent genetic abnormalities in the WHO Classification. It often shows morphology of AML without maturation or with minimal differentiation (some cases described with varied differentiation), with high blast proportions and with a generally poor prognosis. AML with NPM1 mutation • AML with NPM1 mutation is classified within AML with gene mutations in the WHO Classification. It has a relatively good prognosis, usually occurs in patients with normal karyotype with monocytic or myelomonocytic differentiation, occasionally with erythroid differentiation or multilineage dysplasia, has high blast proportions and is often considered to be a primary abnormality Concurrent trisomy 4 and NPM1 • In the few cases described of AML with trisomy 4, NPM1 mutation occurs at a similar frequency as in AML with normal cytogenetics. In one publication, it was most often also associated with FLT3-ITD (Bains). The simultaneous occurrence of trisomy 4 and NPM1 in the current case raises questions of whether either is the primary event or if they are independent genetic abnormalities occurring coincidentally to produce a unique disease. The case initially suggests that in this situation the "worse" genetic feature, trisomy 4, holds sway. This patient initially achieved remission but suffered sustained pancytopenia with possible early relapse after 6 months and has undergone bone marrow transplantation. Karyotypic abnormalities in NPM1 mutated AML • The bulk of available evidence suggests strongly that NPM1 is a “founder” abnormality and that chromosomal abnormalities are secondary events • NPM1 mutation is not found as a secondary event • NPM1 mutation is a stable marker of residual disease • Presence of karyotypic abnormalities, including trisomies of 4, 8 and 11 may not adversely affect prognosis of NPM1 mutated AML • While trisomy 4 has shown concurrent NPM1 mutation, poor prognosis trisomy 11 has not • It has recently been suggested that immune response to mutated NPM1 may contribute to relatively favorable prognosis Clinical Course • The patient was started on induction chemotherapy with Cytarabine 100mg/m2 (milligram per square meter) for 7 days along with Idarubicin 12mg/m2 for 3 days. She had neutropenic fever with E. Coli bacteremia and was treated with antibiotics. Her day 14 bone marrow did not show evidence of leukemia. • After remission was confirmed, she received consolidation therapy with high dose cytarabine. She developed cerebellar toxicity and received only 5 out of 6 planned doses of cytarabine. • Her second consolidation was with Mitoxantrone 10mg/m2 and Etoposide 100mg/m2 daily for 5 days. She developed mucositis and was treated supportively. She remained pancytopenic for 7 weeks. A bone marrow examination was performed. Follow-up marrow • 13.2% blasts/blast-like cells, 15.6% immature monocytoid cells, 13% monocytes and 28% mature/maturing neutrophils. No Auer rods. • Flow Cytometry (blast gate): CD11b, CD13, CD15, CD33, CD64, CD38, partial CD14 and CD41 • Cytogenetics: – Karyotype: 46, XX – FISH: No evidence of trisomy 4, del 5q, 7q or 20q12, or of trisomy 8. • Immunohistochemistry: NPM cytoplasmic positivity in a minority of scattered cells in clot section. • Dx: Normocellular marrow with increased blasts and peripheral pancytopenia, suggesting early relapse of AML but without genetic evidence of recurrence; post-therapy dysplasia also possible. Follow-up marrow Follow-up NPM Further follow-up • She was referred to a transplant center and underwent allogeneic stem cell transplantation with a matched unrelated donor. • She is in bone marrow remission with no cytogenetic or molecular abnormalities. • She is in treatment for GVHD-related complications. Follow-up observations • Post-treatment pancytopenia occurred with apparent early relapse of AML but a smouldering course and different than original phenotype (monocytic, CD34-). • There was loss of trisomy 4, but some residual cells showed cytoplasmic NPM, suggesting that the NPM1 mutation may have been the primary (founder) abnormality, with the disease accelerated or altered by trisomy 4 and persistent at low level after initial therapy. • There was no cytogenetic evidence of post-therapy myeloid malignancy. PROPOSED DIAGNOSIS • Acute Myeloid Leukemia with recurrent genetic abnormality • Current terminology; AML with mutated NPM1, and trisomy 4. • Possible future terminology; AML with trisomy 4, and NPM1 mutation. CONSENSUS DIAGNOSIS • Acute myeloid leukemia, with NPM1 mutation and trisomy 4 References • Haferlach C, et al. AML with mutated NPM1 carrying a normal or aberrant karyotype show overlapping biologic, pathologic, immunophenotypic, and prognostic features. Blood 2009 Oct; 114(14): 3024-3032 • Falini B, et al. Acute myeloid leukemia with mutated nucleophosmin (NPM1): is it a distinct entity? Blood 2011 Jan 27;117(4):1109-20 • Alseraye, et al. Trisomy 11 as an isolated abnormality in acute myeloid leukemia is associated with unfavorable porgnosis but not with an NPM1 or KIT mutation. Int J Clin Exp Pathol 2011;4(4): 371-377 • Bains A, et al. Molecular and clinicopathologic characterization of AML with isolated trisomy 4. Am J Clin Pathol 2012;137:387-394. • Greiner J, et al. Immune responses against the mutated region of cytoplasmic NPM1 might contribute to the favorable clinical outcome of AML patients with NPM1 mutations (NPM1mut). Blood 2013;122(6):1087 • Jain P, et al. Mutated Nucleophosmin-1 (NPM1) in patients with Acute Myeloid Leukemia (AML) in remission and relapse. Leuk Lymphoma 2013 [Epub ahead of print]