Bone Marrow Failure in AML: Residual Neutrophils and Erythroblasts Are Derived By Differentiation from the AML

Our previous work[1] suggests AML blocks the differentiation of normal haematopoietic stem cells (HSCs) leading to marrow failure. However, many patients do not have uniform pancytopenia where all differentiated blood cells are reduced e.g. some patients have preserved neutrophil counts.

We aimed to identify whether the residual mature blood cells at diagnosis of AML are derived from the main AML clone, or from normal HSCs (or pre-leukaemic HSCs) by sequencing highly purified sorted residual neutrophils (CD45+CD13+CD16+) and erythroblasts (CD45-CD71+CD235a+) from 46 fresh AML diagnostic samples using next generation sequencing. T cells and blasts were used as negative and positive controls.

Neutrophils had the same mutational profile as the blasts from 25/45 (55%) cases suggesting that neutrophils were derived by differentiation from the AML clone. No mutations in neutrophils from 8/45 (17%) cases consistent with the neutrophils being derived from normal HSCs. Neutrophils had some but not all the mutations seen in the AML clone from 12/45 (27%) cases; in these cases, neutrophils most often bore mutations in ‘early hit’ genes such as DNMT3A or TET2 suggesting they may have derived from pre-leukaemic HSCs. We did not sequence neutrophils in one case due to insufficient yield of DNA.

Erythroblast numbers were lower, and we were not able to get sufficient numbers to sequence in all AML. The erythroblasts had the same mutational profile as the blasts from 8 of 17 (47%) sequenced cases and some but not all the mutations seen in the AML clone from 5/17 (29%) cases. Mutations were not seen in erythroblasts from 4/17 (24%) cases.

Of 30 cases with preserved neutrophils (>1x10⁹/L) at diagnosis, 20/30 (66%) cases had neutrophils derived from the AML clone, 7/30 (23%) cases had neutrophils derived from pre-leukaemic HSCs and 3/30 (10%) cases had neutrophils derived from normal HSCs. Of the patients with preserved neutrophils, there was a clear association between neutrophil morphology and mutations. Patients with any mutations in the neutrophils (n=27) had neutrophil dysplasia while the 3 cases without mutations in the neutrophils had normal neutrophils morphology.

Of the five of AML cases where the haemoglobin was preserved (>100g/l) at diagnosis, two (40%) cases had erythroblasts derived from the AML clone, one (20%) from pre-leukaemic HSCs and two (40%) cases from normal HSCs.

In conclusion, where neutrophil numbers are preserved at diagnosis, the neutrophils are derived by differentiation of blasts in two-thirds of patients. In one third of cases the neutrophils derive from normal or pre-leukaemic HSCs suggesting the differentiation block we previously identified in normal HSCs may not be complete in all AMLs.

We are testing megakaryocytes using Fluorescence In Situ Hybridization (FISH) to delineate the origin of platelets at diagnosis of AML where the platelets are preserved.

This works helps further define the pathology of marrow failure in AML.

1. Miraki-Moud, F., et al., Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation. Proc Natl Acad Sci U S A, 2013. 110(33): p. 13576-81.

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