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Correlation of mutations with morphological findings in AML
Studies have shown that, although dysplasia is frequently seen in NPM1-mutated AML, it does not confer a worse clinical outcome.8,9 Another study found that in AML patients with wild-type NPM1, those with multilineage dysplasia showed an inferior response to induction and, in younger patients, a lower 5-year survival, suggesting that the prognostic relevance of multilineage dysplasia in AML might depend on NPM1 mutation status.10
In a recent study of patients with de novo AML lacking specific cytogenetic findings, we reported that WHO- defined multilineage dysplasia had no impact on outcome, but the presence of certain specific dysplastic features (micromegakaryocytes and hypogranulated myeloid cells) was associated with adverse EFS.11 However, this study did not evaluate the impact of gene mutations other than FLT3 and NPM1. Moreover, despite the recently expand- ed knowledge of the mutations affecting multiple func- tional pathways in AML, an association of mutations with specific dysplastic features has not been closely evaluated. In myelodysplastic syndromes (MDS), Della Porta et al. found an association between granulocytic dysplasia and mutations in ASXL1, RUNX1, TP53 and SRSF2.12 Devillier et al. evaluated 94 patients with AML-MRC and found that ASXL1 mutations were associated with a higher degree of dysgranulopoiesis, but not dyserythropoiesis or dysmegakaryopoiesis.13 However, this study did not eval- uate individual dysplastic features, and included patients with both MDS-related cytogenetics and a prior history of MDS, who are known to have a poor prognosis and fre- quent ASXL1 mutations.14,15 In AML cases without a histo- ry of MDS or MDS-associated karyotype findings, it is uncertain if the presence of background morphological dysplasia indicates a true relationship to MDS.
The goal of the current study is to analyze the associa- tion between dysplastic findings and somatic mutations in de novo AML. We investigated the associations between specific dysplastic features with individual mutations, gene pathway alterations, and clonal architecture, and explored the effects of these parameters on patients' out- come.
Methods
Patients
Cases of newly diagnosed de novo AML were identified from the pathology archives of Brigham and Women’s Hospital/Dana- Farber Cancer Institute and Massachusetts General Hospital between 2009-2016. This study has been approved by an institu- tional review board (IRB) (IRB Protocol n. 2009P001369). All cases had bone marrow (BM) aspirate smear and biopsy slides available for review that were diagnosed as AML prior to any therapy being administered. Only cases with adequate karyotype and clinical follow-up information were included. Patients who had received any prior cytotoxic therapy, had a prior diagnosis of any myeloid neoplasm, or had defining cytogenetic abnormalities of AML- MRC or AML with recurrent genetic abnormalities according to the 2016 WHO Classification4 were excluded.
Morphology assessment
Bone marrow aspirate and biopsy smears from each case were viewed in a blinded fashion by 3 hematopathologists (OW, RH and OP) who scored dysplasia in each lineage in increments of 10%, as previously described;11 the median score for all 3 observers was used for all analyses. A minimum of 10 megakary-
ocytes (on biopsies and/or aspirate smears) and 20 erythroids and 20 myeloid elements (in aspirate smears) were required, otherwise a lineage was designated as “not evaluable”. Specific dysplastic features in each lineage were also scored on a semi-quantitative scale (<10% cells showing the dysplastic feature = 0, 10-25%=1, 26-50%=2, 51-75%=3, >75%=4). Specific dysplastic features scored in the erythroid lineage were: 1) megaloblastoid change; 2) multinucleation; 3) nuclear irregularities; and 4) pyknosis. Dysgranulopoiesis features scored were: 1) abnormal nuclear shape (including pseudo Pelger-Huet anomaly); and 2) hypogranu- lation. Dysmegakaryopoiesis features scored were: 1) micromegakaryocytes; 2) presence of two or multiple separated nuclear lobes; and 3) megakaryocytes with hypolobated or monolobated nuclei.
Clinical data
The complete blood count (CBC) and white blood cell (WBC) count differential results at the time of AML diagnosis were recorded. Type of treatment, including date of any allogeneic stem cell transplant (SCT), date of relapse or disease refractoriness to two induction regimens, and status at last follow up were record- ed for each patient. Complete remission (CR) was determined as defined by clinical standards13 and follow-up information (relapse, death) was recorded.
Targeted sequencing
We performed targeted sequencing on BM aspirates obtained at the time of diagnosis for all 168 patients, as previously described.16-18 We enriched target regions of 87 genes (Baylor Custom SureSelect hybrid capture system, Agilent Technologies, Santa Clara, CA, USA) in 105 patients or 95 genes [Rapid Heme Panel kit, Illumina Truseq Custom Amplicon (TSCA), San Diego, CA, USA] in 66 patients, which were selected on the basis of path- ogenic involvement in myeloid malignancies, on either banked DNA samples or diagnostic BM aspirate smears. We classified variants as pathogenic driver mutations based on mutation type and position, and on frequency in publicly available single nucleotide polymorphism databases. The median coverage was 1200X across all the genes. Average coverage across the entire hybrid capture run was 500X. The minimal coverage was 50X, or at least 5 alternative reads required to call a variant. Variants with a variant allele fraction of less than 0.05 were excluded to ensure consistency across both sequencing platforms. FLT3-ITD was identified by filtering aligned BAM files for which one side maps to exon 13, 14 or 15 of FLT3 and the other side is unmapped. These one-sided mapped reads were then scanned for the pres- ence of duplications of at least 10 bp. Reads that contained dupli- cations were realigned among themselves to make a final determi- nation of FLT3-ITD status.18
Mutations were grouped into 7 pathways as follows:
DNA methylation: DNMT3A, IDH1, IDH2, and/or TET2 Epigenetic regulators: ASXL1, EZH2, BCOR, SETBP1, BCORL1,
SH2B3, SETD2, and/or CREBBP
Transcription factors: CEBPA (single or double), RUNX1, ETV6,
WT1, and/or PHF6
Cohesin complex: STAG2, PDS5B, RAD21, and/or SMC3 RAS pathway: KRAS, NRAS, ITD, FLT3, KIT, CBL, RIT1,
PTPN11, and/or NF1
Spliceosome pathway: U2AF1, ZRSR2, PRPF40b, SRSF2, and/or
SF3B1
We separately explored the association between morphology and genetics based on AML ontogeny as specified by Lindsley et al.,14 in which the presence of SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 mutations defined secondary type AML, the presence of an NPM1 mutation [unless any second-
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