Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms

Abstract

Key Points• Activating mutations outside exon 10 of MPL were identified in 10% (7 of 69) of triplenegative cases of ET and PMF.• JAK2-V625F and JAK2-F556V were identified in 2 triple-negative cases of ET and were shown to activate JAK-STAT5 signaling.Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. (Blood. 2016;127(3):325-332) IntroductionEssential thrombocythemia (ET), primary myelofibrosis (PMF), and polycythemia vera (PV) are the classical BCR-ABL-negative myeloproliferative neoplasms (MPNs). They are chronic diseases, characterized by clonal expansion of differentiated myeloid cells driven by somatic mutations. Most of the cases are sporadic, however, familial clustering has also been observed. In 95% of PV cases and 50% to 60% of ET and PMF cases the disease is driven by an acquired mutation in the JAK2 gene V617F.1-4 JAK2-V617F leads to the constitutive activation of the Janus kinase 2 (JAK2) and subsequently the JAK-signal transducer and activator of transcription (STAT) signaling pathway. The remaining cases of PV carry mutations in exon 12 of JAK2. Although JAK2 mutations in MPNs are acquired, in recent years several families with hereditary thrombocytosis (HT) have been described to have germline JAK2 mutations. [5][6][7] The second most commonly mutated gene in ET and PMF is CALR encoding calreticulin. Mutations in exon 9 of CALR have been described in 25% and 35% of patients with ET and PMF, respectively. 8,9 There are o...

How I treat essential thrombocythemia

“…Two recent studies have indeed shown that a few triplenegative patients carry activating mutations of MPL outside exon 10, and that these noncanonical mutations may be either inherited or somatically acquired. 23,24 One study has also shown that some patients carry noncanonical, activating mutations of JAK2, which appear to be germ line in most instances. 23 Finally, some patients have evidence of polyclonal hematopoiesis, and most likely do not have a true MPN.…”

“…Hereditary thrombocytosis may be associated with germ line mutations of THPO, the thrombopoietin gene, 63,64 or MPL, the thrombopoietin receptor gene. 23,24,65 Recent reports have described cases of hereditary thrombocytosis associated with noncanonical germ line mutations of JAK2. 23,[66][67][68][69] In familial ET, JAK2 (V617F) is always a somatically acquired event, as in the familial tree reported in Figure 2.…”

“…23,24,65 Recent reports have described cases of hereditary thrombocytosis associated with noncanonical germ line mutations of JAK2. 23,[66][67][68][69] In familial ET, JAK2 (V617F) is always a somatically acquired event, as in the familial tree reported in Figure 2. 34,70,71 In our clinical practice, we interview all patients with thrombocytosis to find out if there is a family history of thrombocytosis or MPN.…”

“…23 Finally, some patients have evidence of polyclonal hematopoiesis, and most likely do not have a true MPN. 23,24 Thus, the socalled triple-negative cases may include patients with ET associated with noncanonical mutations of MPL, individuals with hereditary thrombocytosis (see "Distinguishing familial ET from hereditary thrombocytosis"), and also subjects with nonclonal thrombocytosis (Figure 1). …”

Myeloproliferative neoplasms: A decade of discoveries and treatment advances

“…In other words, 10-15% of patients with PMF or ET do not express any one of the three mutations and are referred to as being "triple-negative" [35]. Most recently, whole exome sequencing revealed the presence of somatic or germline MPL or JAK2 variants in 3 of 8 triple-negative patients with MPN and subsequent MPL/JAK2 entire codon sequencing among 62 ET and 49 PMF triple-negative cases revealed novel somatic or germline MPL variants in 5 of 62 cases and JAK2 variants in 5 of 57 cases [39].…”