Validation of a Novel Clonality Assay in Patients with Clonal Hematologic Disorders

Clonality assays, based on X-chromosome inactivation, discriminate active from inactive alleles. They are useful in the diagnosis and assessment of therapeutic response in clonal hematologic disorders, especially in absence of an identifiable somatic mutations. Skewing of X-chromosome allelic-usage, based on preferential methylation of one of the HUMARA alleles, was reported as evidence of clonal hematopoiesis in ~30% elderly women, precluding the use of this assay in elderly patient (>65 years of age). Using a quantitative, transcriptionally-based clonality assay, we did not detect clonal hematopoiesis in >200 healthy non-elderly women. In view of the susceptibility of aging hematopoietic stem cells to epigenetic dysregulation, we reinvestigated the issue of clonality in forty elderly women (age 65–92, mean 81.3 years), using a novel, quantitative qPCR transcriptional clonality assay. In this assay, mRNA transcribed from five X-chromosome polymorphic genes expressed in peripheral blood neutrophils is quantified by real time, allele specific RT-PCR. We did not detect clonal hematopoiesis in any of the elderly women. However, using HUMARA assay, 30% of these elderly women were detected to have monoallelic methylation of the HUMARA gene locus, consistent with previously reported literature. We concluded that our novel transcriptional clonality assay is suitable for evaluation of clonal hematopoiesis in all women including elderly women (Swierczek S, Agarwal N et al. Blood 2008, July 18 epub). Using this novel assay, we detected clonal hematopoiesis in 31 out of 32 well characterized patients with myeloproliferative disorders:

1. polycythemia vera (all fourteen patients were clonal by our assay and all were JAK2^V617F positive),

2. essential thrombocytosis (nine out of ten patients were clonal by our assay, one out of ten patients was cMPL^W515L positive, seven out of ten were JAK2 ^V617F positive; however one subject with low JAK2 ^V617F allelic burden was polyclonal by our assay), and

3. primary myelofibrosis (all eight patients were clonal by our assay and two of them were positive for JAK2^V617F).

In addition, we detected clonal hematopoiesis in 4 patients with unexplained anemia (two eventually evolved in to myelodysplastic syndrome), and in one patient with persistent leukocytosis (eventually found to be cMPL^W515L positive). Using our assay we did not detect clonal hematopoiesis in 10 patients with reactive or secondary erythrocytosis, thrombocytosis or leukocytosis. We conclude that our novel transcriptional clonality assay is suitable for detection of clonal hematopoiesis in patients with clonal hematologic disorders, especially in patients lacking known somatic mutation. Studies to detect an emerging clone in milieu of polyclonal hematopoiesis (such as seen in PNH or early stages of clonal hematological disorders) by comparison of X-chromosome allelic usage ratio in myeloid cells and in T lymphocytes are underway.