Accumulation of Specific Somatic Leukemia-Associated Mutations in Congenital Neutropenia Precedes Malignant Transformation - New Preconditions for Treatment Decisions

Severe congenital neutropenia (CN) and Shwachman-Diamond Syndrome (SBDS) are pre-leukemia bone marrow failure syndromes with a high cumulative incidence of developing MDS or AML. Clonal hematopoiesis (CH) due to nonsense mutations in the intracellular part of CSF3R or missense TP53 mutations has been reported previously in CN or SBDS patients, respectively. So far, studies of CH in CN were conducted by sequencing CSF3R only (Germeshausen et al., 2007, Klimiankou et al., 2019) or in ELANE-CN patients (Jun Xia et al., 2018). Precise characterization of the timeline and occurrence of somatic mutations found at the neutropenia, pre-leukemia or leukemia stage in CN is essential to make a timely and correct decision on the requirement of bone marrow transplantation prior to transformation to overt leukemia. We therefore aimed to assess the acquisition of somatic leukemia-associated mutations in a large number of genes in a worldwide largest and most representative cohort of CN patients. We performed error-corrected sequencing with fixed single-stranded unique molecular indexes (UMIs) on panel of 31 genes and "hotspot" regions in 340 genes reported to be mutated in de novo MDS/AML. We analyzed 146 CN patients representing most of genetic subtypes of CN, 21 SBDS patients, 29 CN patients who progressed to MDS or AML, and 18 healthy individuals. The median age of CN and SBDS patients was 13,73 and 10,5 years, respectively. For 53 CN patients, we collected sequential samples with a median observation time of 1,1 years.

We found that the prevalence of CH with at least 1 somatic DNA lesions in CN group was 49,5 % which is much higher than in healthy individuals of the same age. The prevalence of CH with CSF3R mutations varied between 20 and 38 % in patients with genetic defects in ELANE (n = 75), HAX1 (n = 26), SRP54 (n = 8), JAGN1 (n = 6), and G6PC3 (n = 5). The percentage of CH positive cases without CSF3R mutations ranged between 12 % and 50 %. CN patients acquired somatic coding mutations most frequently in CSF3R (49 %) following by TP53 (4,1 %), ASXL1 (4,1 %), DNMT3A (2,1 %), TET2 (2,1 %), JAK2 (1,37 %), SETBP1 (1,37 %), ARID1B (1,37 %), GNAS (1,37 %), and ZRSR2 (1,37 %). Interestingly, in the SRP54-CN group, we found 1 patient with CSF3R mutation, 1 patient with TP53 mutations, and 1 patient with both mutations. Out of 3 CLPB-CN patients, 1 patient has CSF3R and 1 patient acquired TP53 mutations.

High prevalence of CSF3R (67 %), RUNX1 (44 %), SETBP1 (22 %), ASXL1 (17 %), ZRSR2 (11 %), NPM1 (11 %) and PTPN11 (11 %) mutations was detected in CN patients with overt MDS/AML, arguing for a specific pathway which initiates and drives leukemic transformation in CN. Comparison of CN patients who later developed MDS/AML with MDS/AML-free CN cohort revealed enrichment of CSF3R (Fisher's exact test; OR 3,42; 95% CI, 1,27, - 9,36, p = 0,01), and ASXL1 (Fisher's exact test; OR 7,4; 95% CI, 0,93 - 59,16, p = 0,03) mutations. In contrast, RUNX1 and SETBP1 mutations were associated with overt MDS/AML in CN. These data argue that the acquisition of CSF3R and ASXL1 mutations are early events in CN leukemogenesis, whereas RUNX1 and SETBP1 mutations may have stronger leukemogenic potential and therefore are rarely found at the neutropenia stage. Moreover, mutation burden at the neutropenia stage in CN patients who later progressed to MDS/AML was significantly higher (p = 0.016, by Fisher's exact test), as compared to CN group who haven't developed leukemia so far. The number of mutations in samples prior AML/MDS was dependent on duration to overt AML/MDS with gradual accumulation of genetic lesions over time.

CH was detected in 66,7 % of SBDS patients with a high prevalence of TP53 mutations (9 out of 14 patients with CH) followed by missense mutations in IDH1 (n = 1), FOXP1 (n = 1), and PTPN11 (n = 1). Our results are in line with previous publications about dominant role of TP53 mutation in SBDS patients.

Prevalence of CH didn't differ between CN patients depending on G-CSF dose (median = 4,06 μg/kg/d) in two biggest CN cohorts harboring ELANE or HAX1 mutations.

Taken together, CN per se contributes to high level of CH already in the young age, as compared to healthy population. Acquisition of multiple genetic lesions, especially RUNX1, SETBP1, ASXL1, TP53, PTPN11 in association with CSF3R mutation might be a strong indicator of the advanced pre-leukemia stage in CN patients at neutropenia stage and requires closer patient follow up.

Dale:X4 Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.