Next-generation sequencing techniques have revealed that leukemic cells in acute myeloid leukemia often are characterized by a limited number of somatic mutations. These mutations can be the basis for the detection of leukemic cells in follow-up samples. The aim of this study was to identify leukemia-specific mutations in cells from patients with acute myeloid leukemia and to use these mutations as markers for minimal residual disease. Leukemic cells and normal lymphocytes were simultaneously isolated at diagnosis from 17 patients with acute myeloid leukemia using fluorescence-activated cell sorting. Exome sequencing of these cells identified 240 leukemia-specific single nucleotide variations and 22 small insertions and deletions. Based on estimated allele frequencies and their accuracies, 191 of these mutations qualified as candidates for minimal residual disease analysis. Targeted deep sequencing with a significance threshold of 0.027% for single nucleotide variations and 0.006% for NPM1 type A mutation was developed for quantification of minimal residual disease. When tested on follow-up samples from a patient with acute myeloid leukemia, targeted deep sequencing of single nucleotide variations as well as NPM1 was more sensitive than minimal residual disease quantification with multiparameter flow cytometry. In conclusion, we here describe how exome sequencing can be used for identification of leukemia-specific mutations in samples already at diagnosis of acute myeloid leukemia. We also show that targeted deep sequencing of such mutations, including single nucleotide variations, can be used for high-sensitivity quantification of minimal residual disease in a patient-tailored manner.

SummaryB-1 lymphocytes produce natural immunoglobulin (Ig)M, among which a large proportion is directed against apoptotic cells and altered self-antigens, such as modified low-density lipoprotein (LDL). Thereby, natural IgM maintains homeostasis in the body and is also protective against atherosclerosis. Diabetic patients have an increased risk of developing certain infections as well as atherosclerosis compared with healthy subjects, but the underlying reason is not known. The aim of this study was to investigate whether diabetes and insulin resistance affects B-1 lymphocytes and their production of natural IgM. We found that diabetic db/db mice had lower levels of perito-

Mutations in NPM1 can be used for minimal residual disease (MRD) analysis in acute myeloid leukemia (AML). We here applied a newly introduced method, deep sequencing, allowing for simultaneous analysis of all recurrent NPM1 insertions and thus constituting an attractive alternative to multiple PCRs for the clinical laboratory. We retrospectively used deep sequencing for measurement of MRD pre- and post-allogeneic hematopoietic stem cell transplantation (alloHCT). For 29 patients in morphological remission at the time of alloHCT, the effect of deep sequencing MRD on outcome was assessed. MRD positivity was defined as variant allele frequency ≥0.02%. Post-transplant MRD status was significantly and independently associated with clinical outcome; 3-year relapse-free survival 20% vs 85% (p < .001), HR 45 (95% CI 2-1260), and overall survival 20% vs 89% (p < .001), HR 49 (95% CI 2-1253). Thus, the new methodology deep sequencing is an applicable and predictive tool for MRD assessment in AML.

Background. Lipolysis of lipoproteins by secretory phospholipase A 2 group V (sPLA 2 -V) promotes inflammation, lipoprotein aggregation and foam cell formation -all considered as atherogenic mechanisms.

CME Accreditation Statement: This activity ("JMD 2019 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("JMD 2019 CME Program in Molecular Diagnostics") for a maximum of 18.0 AMA PRA Category 1 Credit(s) ä. Physicians should claim only credit commensurate with the extent of their participation in the activity.