Clonal Hematopoiesis in Philadelphia Chromosome-Negative Bone Marrow Cells of Chronic Myeloid Leukemia Patients Receiving Tyrosine Kinase Inhibitors
Authors: Paquette Ron , Nicoll John , Chalukya Meenal , Gondek Lukasz P , Jasek Monika , Sawyers Charles , Shah Neil , Maciejewski Jaroslaw 
Published: April 2020

Abstract
Patients with chronic myeloid leukemia (CML) are experiencing prolonged survival due to successful therapy with tyrosine kinase inhibitors. However, some CML patients who have achieved longstanding remissions with these agents harbor clonal cytogenetic abnormalities in their Philadelphia chromosome negative (Ph-) bone marrow cells. Because CML patients in remission often have peripheral blood count abnormalities, including cytopenias, we investigated whether these patients may have developed myelodysplastic syndrome (MDS) within the Ph- cell population. Bone marrow samples from 26 CML patients who had achieved a major cytogenetic remission (MCyR) with tyrosine kinase inhibitor therapy between 2 and 15 years after diagnosis were evaluated; 6 patients had advanced disease prior to their last therapy, 20 were in chronic phase. At the time of evaluation, 2 of the patients were receiving imatinib, 23 dasatinib, and 1 PHA739358. At least one peripheral blood lineage was abnormal in 21 patients, of whom 7 had pancytopenia. Routine metaphase cytogenetics (MC) revealed a persistent clonal chromosomal abnormality in 10% of the Ph- metaphases in 5 patients (+8 in 2, −7 in 2, and 20q- in 1). We hypothesized that clonal hematopoiesis might exist in additional patients and applied single nucleotide array (SNP-A) based karyotyping and X-linked human androgen receptor (HUMARA) clonality assay to further delineate the nature of the hematopoietic defect in these patients. HUMARA was performed on bone marrow samples and germ-line DNA from peripheral blood T lymphocytes of the female patients. Clonality, as assessed by skewing of X-chromosome inactivation in bone marrow cells compared to germline control cells, could not be demonstrated in the12 female patients. SNP-A karyptyping using 250K Affymetrix SNP array confirmed the known cytogenetic abnormalities. Several microdeletions were found, but comparison with purified T lymphocytes demonstrated that these “lesions” represented germ line-encoded copy number variants. However, SNP-A karyotyping revealed the presence of uniparental disomy (UPD) involving chromosome 17(p12-pter) in bone marrow, but not germ line cells, from one male patient with normal karyotype by routine MC. In the context of secondary AML, del17p or UPD17 have been observed always in the presence of del7/q and 5q and were associated with poor prognosis. However, in our patient UPD17 occurred as a sole defect. Because in our studies in AML, UPD of chromosome 17p was found in association with p53 mutations, genomic sequencing of this gene was performed. A 5 bp deletion destroying the splice acceptor region of exon 6 was identified in bone marrow cells from this patient. Alternative splicing leading to loss of exon 6 was predicted to result in a frame shift and premature introduction of a stop codon. These methods revealed clonal hematopoiesis in the Ph- bone marrow cells of 6/26 patients with longstanding CML in remission from tyrosine kinase inhibitors and persistent peripheral blood abnormalities. The approaches used here probably underestimate the frequency of this condition, as oligoclonal populations may be present in numbers below the limit of assay sensitivity. The Ph- clonal bone marrow populations have cytogenetic and molecular features in common with MDS. After a median follow up of two years, one patient with monosomy 7 developed acute myeloid leukemia, but longer follow up will be required to determine the natural history of the Ph- clonal disorders.