CD69 Is a Very Useful Marker for Minimal Residual Disease and Related Mast Cell Assessment of Acute Myeloid Leukemia

background：Minimal residual disease (MRD) assessment using multicolor flow

cytometry (MFC) has become the center point of acute myeloid leukemia (AML) risk stratification and therapeutic management. The addition of new markers can improve the accuracy and applicability of MFC based MRD assay further because of the complexity and heterogeneity of malignant myeloblasts，especially genetic related markers. Besides，some studies had testified by flow cytometry sorting (FACSorting) that the fusion gene existed in mast cells in a subgroup of AML patients who had positive RUNX1-RUNXT1 genes in complete response (CR) period. This method was time-consuming and expensive, it would be more feasible and beneficial to the fellow-up of this kind of patients if we could differentiate genetic normal or abnormal mast cells by CD69 and other markers' expression. Herein, we evaluated the utility of a new marker, CD69 in the assessment of MFC-based MRD and mast cells.

Methods Expression patterns and intensity of CD69 were studied using 8 or 9-color MFC in leukemic blasts from bone marrow (BM) samples of 121 AML patients, which including initial diagnostic and MRD samples. Normal myeloblasts from BM samples of 30 CR patients with unrelated diseases were taken as normal controls. To quantitatively study the intensity of CD69 expression, the mean fluorescence intensity (MFI) of CD69 in CD69 negative lymphocytes was taken as a corrective control, the MFI ratio (MFIR) was calculated by dividing the MFI of CD69 in normal or malignant myeloblasts by that of corrective control. 52 kinds of common fusion genes and 58 kinds of gene mutation related to AML were monitored at the same time for 81 patients by real-time quantitative polymerase chain reaction (RT-PCR) and 77 cases by next generation sequencing (NGS) , as well as chromosome examination for 79 cases, aiming to find the relationship of CD69 expression with genetics. The same method was taken to study the CD69 expression in mast cells from BM samples of 20 genetic negative CR AML patients and 3 RUNX1-RUNXT1 fusion gene positive CR AML patients. The expression of CD2, CD25 and other myeloid markers were detected to establish a normal expression pattern of related markers on benign mast cells with aim to identify the abnormal mast cells by different from normal (DFN). For the three RUNX1-RUNXT1 positive CR patients, CD34 and CD117 double positive myeloblasts and mast cells were sorted by FACSAria MFC and RUNX1-RUNXT1 fusion gene was detected respectively.

Results CD69 was negative in normal myeloblasts. In malignant myeloblasts of 121 AML cases, the positive or partially positive rate of CD69 was 42.15%(51/121 cases). The MFIR was 10.45（1.58-29.31）, significantly higher than that of the control group (P < 0.01). The expression of CD69 was positively related to DNMT3A gene mutation, and negatively related to Kit and CEBPA mutation. No relationship was found between CD69 expression and fusion genes or abnormal karyotype. The MFIR of CD69 was 0.51（0.10-0.89）for normal mast cells from genetic negative controls. The expression of CD69 was obviously brighter on the mast cells from three RUNX1-RUNXT1 positive patients, with high expression of CD33 and abnormal expression of CD2 and CD25. It was testified that RUNX1-RUNXT1 located in abnormal mast cells rather than in myeloblasts by FACSoring and gene detection.

Conclusion CD69 was an effective AML MRD marker for the high positive rate and specificity and might become a promising target for chimeric antigen receptor-modified T(CAR-T) cells therapy to AML. Establishing a reference range of CD69 expression on normal myeloblasts and mast cells as MFIR, might be applied to effectively recognize malignant myeloblasts and abnormal mast cells. Especially for recently identified abnormal mast cells who harbored RUNX1-RUNXT1 fusion gene, detecting CD69 and related markers expression on mast cells by MFC would be a highly cost-effective method comparing to FACSorting combing gene examination. This would play an important role in next step research and offering guidance to clinical therapy.