Illumina MiSeq paired end sequencing; DNA-Seq of human Acute Panmyelosis with Myelofibrosis (APMF) Samples
DNA-Seq of human Acute Panmyelosis with Myelofibrosis (APMF) Samples
We searched the archives of the Institute of Surgical Pathology of the University Medical Center, Albert-Ludwigs-University Freiburg for formalin-fixed and paraffin-embedded (FFPE) bone marrow (BM) core biopsies from de novo acute myeloid leukemia (AML) cases with BM fibrosis at least grade 2 on a 0 to 3 scale diagnosed between 1997 and 2013. Clinical, haematological and survival data were obtained from medical records. Cases with a history of myelodysplastic syndrome, myeloproliferative neoplasm, previous malignancies, autoimmune disorders or exposure to chemotherapy, irradiation or toxic substances were excluded. Histologic and immunohistologic slides of the BM specimens were reviewed by 2 investigators (A. S-G, S.K-W). We strictly applied the clinicopathologic diagnostic criteria of the revised 2016 WHO classification of myeloid disorders for the diagnosis of acute panmyelosis with myelofibrosis (APMF): acute onset of disease, no/or minimal splenomegaly, percentage of BM  and/or peripheral blood blasts ≥ 20%, no teardrop-shaped peripheral blood cells, no reported cytogenetic or molecular abnormalities characteristic for any other WHO-defined AML entity, BM fibrosis ≥ grade 2, panmyelosis showing increased precursors of all three cell lineages. On the basis of these criteria, we identified 16 APMF patients whose FFPE BM core biopsies could be used for further molecular investigations. No fresh frozen blood, BM samples or germ line controls were available from this cohort. 5 Samples yielded sufficient quantity and/or quality for NGS analysis. Deparaffinization and DNA extraction were performed using the Qiagen QiaAMP DNA extraction kit for FFPE tissue (Qiagen, Valencia, CA) according to manufacturer's instructions and quantified using the Qubit fluorometric assay (Thermo Fisher Scientific, Foster City, CA).  Illumina TruSight Myeloid and Tumor 15 NGS panels were processed as described in the manufacturers instructions. For both panels, the maximal possible volume of DNA was used for library construction.
Experimental Factor: individual
13