Discrimination of polycythemias and thrombocytoses by novel, simple, accurate clonality assays and comparison with PRV-1 expression and BFU-E response to erythropoietin

Abstract

Essential thrombocythemia (ET) and polycythemia vera (PV) are clonal myeloproliferative disorders that are often difficult to distinguish from other causes of elevated blood cell counts. Assays that could reliably detect clonal hematopoiesis would therefore be extremely valuable for diagnosis. We previously reported 3 X-chromosome transcription-based clonality assays (TCAs) involving the G6PD, IDS, and MPP1 genes, which together were informative in about 65% of female subjects. To increase our ability to detect clonality, we developed simple TCA for detecting the transcripts of 2 additional X-chromosome genes: Bruton tyrosine kinase (BTK) and 4-and-a-half LIM domain 1 (FHL1). The combination of TCA established the presence or absence of clonal hematopoiesis in about 90% of female subjects. We show that both genes are subject to X-chromosome inactivation and are polymorphic in all major US ethnic groups. The 5 TCAs were used to examine clonality in 46 female patients along with assays for erythropoietin-independent erythroid colonies (EECs) and granulocyte PRV-1 mRNA levels to discriminate polycythemias and thrombocytoses. Of these, all 19 patients with familial polycythemia or thrombocytosis had polyclonal hematopoiesis, whereas 22 of 26 patients with clinical evidence of myeloproliferative disorder and 1 patient with clinically obscure polycythemia were clonal. Interestingly, interferon alpha therapy in 2 patients with PV was associated with reversion of clonal to polyclonal hematopoiesis. EECs were observed in 14 of 14 patients with PV and 4 of 12 with ET, and increased granulocyte PRV-1 mRNA levels were found in 9 of 13 patients with PV and 2 of 12 with ET. Thus, these novel clonality assays are useful in the diagnosis and follow-up of polycythemic conditions and disorders with increased platelet levels.

Interferon-α therapy in bcr-abl-negative myeloproliferative neoplasms

“…This possibly selective effect on the MPN clone is further supported by several in vivo studies showing reversion from monoclonal to polyclonal patterns of hematopoiesis (based on X-chromosome inactivation pattern studies) or disappearance of a chromosomal abnormality present before treatment in IFN-a-treated patients. [18][19][20][21] Using quantification of JAK2V617F mutated alleles in circulating granulocytes as a marker of minimal residual disease, our group could also show that IFN-a was able to markedly decrease the proportion of circulating mutated cells. 22,23 The megakaryocytic lineage seems particularly sensitive to IFN-a, with clear morphological and biochemical changes of megakaryocytes in IFN-treated patients.…”

“…The situation may change with the development of JAK2 inhibitors, 83 but to date only IFN-a has been able to induce cytogenetic remissions or reversion from monoclonal to polyclonal hematopoiesis in some patients, [18][19][20][21] suggesting that it could eradicate the malignant clone and possibly cure the disease in selected cases. By monitoring the JAK2V617F mutation in PV patients treated with peg-IFNa-2a, we also showed that the V617F mutation could be reduced to undetectable levels (using a PCR assay with 1% sensitivity) in circulating granulocytes in 24% of patients ('molecular remission').…”

Genetic complexity of myeloproliferative neoplasms

“…33,55 The presence of clonal hematopoiesis has been a fundamental part of MPN definition and played an important role in distinguishing MPN from reactive conditions such as secondary polycythemia or reactive thrombocythemia. The discovery of polyclonal ET 56,57 initially raised questions on the diagnostic criteria applied for ET and suggested the genetic heterogeneity of this MPN entity. Before the discovery of JAK2 mutations, polyclonal ET was suspected to represent a reactive condition mimicking ET.…”

Clonal granulocytes in polycythaemia vera and essential thrombocythaemia have shortened telomeres

“…Several studies however have shown that a sizeable proportion of patients with PV and ET have polyclonal granulopoiesis (Ferraris et al, 1999;Harrison et al, 1999;Liu et al, 2003). Our investigation was focused on PV and ET, and as we chose to exploit XCIP as marker of clonality, we found a significant correlation between monoclonal expression of XCIP and shorter telomere lengths in cells of myeloid origin; the seven clinically undistinguishable patients with polyclonal granulopoiesis were found to express a DTRF well within the control range (Fig 1).…”

“…Without the aid of a specific cytogenetic marker, clonality studies have relied mainly on the analysis of X chromosome-linked polymorphic genes, whose expression is subject to random inactivation in females. Contrary to the general principle that PV and ET are monoclonal disorders, several studies have lately demonstrated through analysis of X chromosome inactivation pattern (XCIP) the heterogeneity of clonal development of myeloid cells in CMPDs other than chronic myelogenous leukaemia (CML) (Ferraris et al, 1999;Harrison et al, 1999;Liu et al, 2003).…”