Malignant Transformation in Relationship to Clonal Hematopoiesis in CIN

Background: Chronic Idiopathic Neutropenia (CIN) is a neutrophil disorder characterized by the persistent and unexplained reduction in the number of peripheral blood (PB) neutrophils. The diagnostic criteria for CIN largely overlap with the idiopathic cytopenia of undetermined significance (ICUS)-Neutropenia proposed criteria. We have recently performed NGS analysis of myeloid genes for identification of clonal hematopoiesis (CH) in a cohort of well characterized CIN patients (n=257; aged 20-85 years, median 51 years, 54 males, 203 females). CH was identified in 33/257 patients (i.e. 12.8%).

Aims: To assess CH mutations at baseline and malignant transformation timepoints in our cohort of CIN patients in order to associate the clinical significance of detected clonal aberrations with disease evolution to hematological neoplasm.

Methods: Genomic DNA was extracted from bone marrow or peripheral blood samples at baseline and follow-up timepoints, sequencing libraries were prepared and subjected to targeted NGS on an NextSeq2000 Sequencer (Illumina) using a panel of myeloid genes recurrently mutated in hematological malignancies.

Results: The frequency of transformation to a hematological malignancy was low in the total group of patients (6/257 CIN patients i.e. 2.33%). Of the transformed patients, 5 belonged to the clonal (5/33 clonal patients, i.e. 15.15%) and one to the non-clonal (1/224 non-clonal patients, i.e. 0.44%) group (P<0.0001). The presence of CH conferred a relative risk (RR) 33.94 (95% confidence intervals 5.362 - 214.4; P<0.0001) for malignant transformation. The median time between baseline and malignant progression was 18 months (range 13-169) whereas the median age at baseline was 64 years (range 51-67) and at transformation was 68 years (range 52-82). The median number of mutations per patient was 2 (range 1-3) at baseline and 4 (range 2-5) at transformation. The spectrum of hematological neoplasms included chronic myelomonocytic leukemia (CMML) (n=2), unclassifiable MDS/myeloproliferative neoplasm (MDS/MPN) (n=1), acute (myelomonocytic) myeloid leukemia (AML) (n=1), MDS with multilineage dysplasia (n=1; non-clonal patient) and T-acute lymphoblastic leukemia (T-ALL) (n=1). Patient (#1), who progressed to AML, carried an IDH1 p.R132S mutation (VAF 12.75%) at baseline. At the time of progression he acquired the typical NPM1 p.L287fs mutation (VAF 36.24%; 15 months after the identification of the first clone) with concomitant expansion of the IDH1 clone (VAF 48.73%). Patient (#2), who transformed to MDS/MPN, acquired two novel mutations in JAK2 p.V617F (VAF 33.94%) and ASXL1 p.S747fs (VAF 34.72%) 18 months after the identification of the initial clones (IDH2 p.R140Q, VAF 23.19% and SRSF2 p.P95R, VAF 25.5%). At the time of progression, the size of the baseline clones increased to VAF 35.11% and VAF 35.34%, respectively. Patient (#3) who transformed to T-ALL acquired a mutation in ETV6 p.E100fs (VAF 9.61%) at the time of leukemic transformation, 13 months after the identification of the baseline clones (DNMT3A p.R736H, VAF 24.55%, DNMT3A p.Q248fs 17.3% and IDH1 p.R132S, VAF 20.90%). Clonal expansion of the baseline clones was also observed at the time of progression. Patient (#4) acquired a mutation in TET2 p.K580fs (VAF 29.63%) 40 months after the identification of the first clone (SRSF2 p.P95R, VAF 27.6%). After 140 months the patient progressed to CMML and mutational screening showed increase of both mutated clones to almost VAF 50% and acquisition of a KRAS p.Q61R mutation (VAF 47.79%). The fifth patient (#5) who progressed to CMML carried a double TET2 mutation at baseline (TET2 p.Q969* VAF 8.67% and TET2 p.H1382R VAF 13.94% respectively). At the time of progression, after 169 months, the patient acquired 3 additional mutations: RUNX1 p.T188I VAF 30.9%, SRSF2 p.P95H VAF 30.4% and NRAS p.G12D VAF 2.2% with concomitant expansion of the baseline clones.

Conclusions: CIN patients with CH have increased propensity to develop overt MDS/AML. All transformed patients displayed clonal expansion as was reflected by the increase of VAF and acquisition of additional novel mutations at the time of progression. This ongoing study of sequential NGS analysis of CH in CIN patients is anticipated to enrich further the knowledge on the natural history of this rare disease as well as on the role of CH in clonal evolution toward leukemia.