Additional Genetic Alterations and Clonal Evolution of MPNs with Double Mutations on the MPL Gene: Two Case Reports

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are two of the main BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs) characterized by abnormal megakaryocytic proliferation. Janus kinase 2 (JAK2) mutations are detected in 50–60% of ET and PMF, while myeloproliferative leukemia (MPL) virus oncogene mutations are present in 3–5% of cases. While Sanger sequencing is a valuable diagnostic tool to discriminate the most common MPN mutations, next-generation sequencing (NGS) is a more sensitive technology that also identifies concurrent genetic alterations. In this report, we describe two MPN patients with simultaneous double MPL mutations: a woman with ET presenting both MPLV501A-W515R and JAK2V617F mutations and a man with PMF displaying an uncommon double MPLV501A-W515L. Using colony-forming assays and NGS analyses, we define the origin and mutational landscape of these two unusual malignancies and uncover further gene alterations that may contribute to the pathogenesis of ET and PMF.

JAK2 is a non-receptor tyrosine kinase that contributes to myeloid cell proliferation and differentiation [4,5]. When JAK2 displays the common V617F mutation, the ensuing protein acquires constitutive catalytic activity even in the absence of cytokine stimulation [6]. CALR is an endoplasmic reticulum chaperone protein. The main mutations consist of insertions or deletions in exon 9, resulting in a positively charged C-terminus. The mutant CALR forms a stable complex with the thrombopoietin receptor (TPO-R) upregulating the JAK/STAT pathway [7]. The MPL gene encodes for TPO-R. Excessive TPO-R activation promotes megakaryocytic hyperplasia and bone marrow fibrosis [8,9]. The most frequently reported MPL mutation involves the W515 residue on exon 10 [10].
Double MPL mutations have been previously described but represent an extremely infrequent event [11][12][13][14][15][16]. Ma et al. identified an MPL W515L mutation associated with the S505N substitution [12,13]. Pietra and colleagues detected double MPL mutations in three subjects: two ET patients, one presenting the S505C-W515L double mutation and the other with the V501A-W515R sequence alteration, and an individual with PMF displaying the V501A-W515L mutations [11]. However, the presence of additional genetic alterations possibly contributing to disease initiation and progression was not investigated [16][17][18]. Furthermore, these reports failed to establish if the observed genetic alterations were in cis (on the same allele) or in trans (on two different alleles) [11,19].
In this report, we describe two patients diagnosed with MPNs expressing two concurrent MPL mutations identified by Next Generation Sequencing (NGS) that were investigated for the presence of additional genetic alterations in order to discern the possible clonal evolution of their disease.

Case 1
In May 2017, a 57-year-old woman presenting ischemic symptoms was admitted to the Hematology Unit of the A.O.U. Policlinico "G. Rodolico-San Marco" in Catania with a high platelet count (807 × 10 9 /L) ( Table 1).  [20], the patient was assessed for the presence of JAK2 and CALR mutations by Sanger sequencing (SS). In particular, the genomic DNA (gDNA) was extracted from 1.7 mL of peripheral blood (PB) using the Qiasymphony DSP DNA Midi kit (Qiagen), according to the manufacturer's protocol. For both analyses, we performed a Polymerase Chain Reaction (PCR) using 300 ng of gDNA. To amplify exon 14 of JAK2, we employed the forward (FW) primer 5 -GGGTTTCCTCAGAACGTTGA-3 and the reverse (RV) primer 5 -TCATTGCTTTCCTTT TTCACAA-3 . To amplify exon 9 of CALR, we employed the FW 5 -CCTGCAGGCAGCAG AGAAAC-3 and the RV 5 -ACAGAGACATTATTTGGCGCG-3 primers. The PCR conditions consist of an initial denaturation step at 95 • C for 5 min, then 35 cycles of denaturation (95 • C for 45 s), annealing (57 • C for 30 s for JAK2; 56.5 • C for 30 s for CALR), extension (72 • C for 30 s), and a final extension at 72 • C for 7 min. The final PCR products (460 bp for JAK2 and 288 bp for CALR) were loaded on an agarose gel in TAE 1.2%, purified, and sequenced by SS using the same FW primers employed for the PCR. Both sequencing results were negative for the presence of mutations in the two genes.
Subsequently, we tested the gDNA of the patient for the presence of MPL mutations on exon 10. In particular, 700 ng of gDNA were employed to perform a PCR using the FW 5 -AGTAGGGGCTGGCTGGATGA-3 and the RV 5 -TGCCTGTTTACAGGCCTTCG-3 primers. The Hot Start PCR conditions consist of an initial denaturation step at 94 • C for 5 min, then 80 • C for 1 min, followed by 35 cycles of denaturation (94 • C for 30 s), annealing (60.2 • C for 50 s), extension (72 • C for 30 s), and a final extension at 72 • C for 10 min. The final PCR product of 235 bp was loaded on an agarose gel in TAE 1.2%, purified, and sequenced by SS using the same FW primer employed for the PCR. Sequencing of the MPL gene revealed a V501A (c.1502T>C) substitution ( Figure 1). annealing (60.2 °C for 50 s), extension (72 °C for 30 s), and a final extension at 72 °C for 10 min. The final PCR product of 235 bp was loaded on an agarose gel in TAE 1.2%, purified, and sequenced by SS using the same FW primer employed for the PCR. Sequencing of the MPL gene revealed a V501A (c.1502T>C) substitution ( Figure 1).
The patient underwent a bone marrow (BM) aspiration that revealed megakaryocytic hyperplasia and grade 1 fibrosis. Eight months later (February 2018), she still presented a high platelet count (931 × 10 9 /L). A second BM biopsy confirmed her ET diagnosis. Spleen size was normal. To better analyze the molecular profile of this patient, we performed an NGS analysis. The sensitivity of NGS is much greater than that of SS, as it is able to detect mutant alleles with a frequency lower than 1%, while SS has a limit of detection around 20%. We analyzed the DNA isolated from the patient's PB and BM, employing the Ion Ampliseq Cancer Hotspot Panel v2 (Thermo Fisher Scientific, Waltham, MA, USA) as previously reported [21]. This NGS panel consists of 207 primer pairs covering approximately 2.800 COSMIC (Catalogue of Somatic Mutations in Cancer) mutations from 50 oncogenes and tumor suppressor genes. Sequences were analyzed with the Ion Reporter software version 5.16 (Thermo Fisher Scientific). Variants with coverage lower than 100× and an allele frequency (AF) lower than 5% were filtered out. In the PB sample, our analysis identified both the V501A and the W515R mutations, the latter not detected by SS since it was expressed with a variant allele frequency (VAF) < 20% (14.36%) ( Table 2). The NGS analyses performed on the BM confirmed both MPL mutations with a VAF of 100%. We also detected 5 additional hotspot mutations: JAK2 V617F (VAF 39.77%), PTEN Q17H (VAF 27.88%), TP53 H178Tfs*69 (VAF 11.5%), PIK3CA N1044D (VAF 6.02%), and KIT N566T (VAF 1.62%) ( Table 2). The relatively low VAF of the JAK2 mutation (39.77%) might explain why this alteration was not detected in her PB, as it may have been expressed in a limited number of neoplastic clones in the PB.  The patient underwent a bone marrow (BM) aspiration that revealed megakaryocytic hyperplasia and grade 1 fibrosis. Eight months later (February 2018), she still presented a high platelet count (931 × 10 9 /L). A second BM biopsy confirmed her ET diagnosis. Spleen size was normal.
To better analyze the molecular profile of this patient, we performed an NGS analysis. The sensitivity of NGS is much greater than that of SS, as it is able to detect mutant alleles with a frequency lower than 1%, while SS has a limit of detection around 20%. We analyzed the DNA isolated from the patient's PB and BM, employing the Ion Ampliseq Cancer Hotspot Panel v2 (Thermo Fisher Scientific, Waltham, MA, USA) as previously reported [21]. This NGS panel consists of 207 primer pairs covering approximately 2.800 COSMIC (Catalogue of Somatic Mutations in Cancer) mutations from 50 oncogenes and tumor suppressor genes. Sequences were analyzed with the Ion Reporter software version 5.16 (Thermo Fisher Scientific). Variants with coverage lower than 100× and an allele frequency (AF) lower than 5% were filtered out. In the PB sample, our analysis identified both the V501A and the W515R mutations, the latter not detected by SS since it was expressed with a variant allele frequency (VAF) < 20% (14.36%) ( Table 2). The NGS analyses performed on the BM confirmed both MPL mutations with a VAF of 100%. We also detected 5 additional hotspot mutations: JAK2 V617F (VAF 39.77%), PTEN Q17H (VAF 27.88%), TP53 H178Tfs*69 (VAF 11.5%), PIK3CA N1044D (VAF 6.02%), and KIT N566T (VAF 1.62%) ( Table 2). The relatively low VAF of the JAK2 mutation (39.77%) might explain why this alteration was not detected in her PB, as it may have been expressed in a limited number of neoplastic clones in the PB.

Case 2
In October 2018, a 68-year-old man was referred to the Hematology Unit of the A.O.U. Policlinico "G. Rodolico-San Marco" in Catania with high white blood cell (102 × 10 9 /L) and platelet (785 × 10 9 /L) counts (Table 3). He was promptly subjected to mutational screening by SS for the expression of the JAK2 V617F substitution, but the test was negative as it was for CALR mutations. Subsequent analyses for additional genetic alterations detected the presence of two mutations on the MPL gene: V501A (c.1502T>C) and W515L (c.1544G>T) (Figure 2a). The BM biopsy showed granulocyte hyperplasia and megakaryocyte clustering with moderate (grade 1) fibrosis, and, on this basis, the diagnosis of early PMF was formulated. A mild splenomegaly was detected (longitudinal diameter: 13 cm).

Case 2
In October 2018, a 68-year-old man was referred to the Hematology Unit of the A.O.U. Policlinico "G. Rodolico-San Marco" in Catania with high white blood cell (102 × 10 9 /L) and platelet (785 × 10 9 /L) counts (Table 3). He was promptly subjected to mutational screening by SS for the expression of the JAK2 V617F substitution, but the test was negative as it was for CALR mutations. Subsequent analyses for additional genetic alterations detected the presence of two mutations on the MPL gene: V501A (c.1502T>C) and W515L (c.1544G>T) (Figure 2a). The BM biopsy showed granulocyte hyperplasia and megakaryocyte clustering with moderate (grade 1) fibrosis, and, on this basis, the diagnosis of early PMF was formulated. A mild splenomegaly was detected (longitudinal diameter: 13 cm). To establish if the detected double mutations were in cis or in trans, we carried out the clonal selection of Burst-Forming Units-Erythroid (BFU-E) and Colony-Forming Units-Granulocyte and Monocyte (CFU-GM), followed by SS on the DNA extracted from To establish if the detected double mutations were in cis or in trans, we carried out the clonal selection of Burst-Forming Units-Erythroid (BFU-E) and Colony-Forming Units-Granulocyte and Monocyte (CFU-GM), followed by SS on the DNA extracted from these colonies. In detail, 1 × 10 5 primary mononuclear cells (MNCs) were isolated from BM biopsies by density gradient centrifugation. For the colony-forming assay, MNCs were plated at a density of 10.000 cells/mL (in triplicate) in Methocult H4435 (StemCell Technologies, Vancouver, B.C., Canada) and incubated at 37 • C in 5% CO 2 for 12 days. Forty individual colonies (20 BFU-E and 20 CFU-GM) were plucked and transferred into PCR tubes for DNA extraction by thermic lysing (94 • for 10 min). Subsequently, DNA was subjected to the same PCR described above for the amplification of MPL exon 10 and sequenced by SS [22]. Twelve out of 20 BFU-E colonies carried the heterozygous cis V501A-W515L mutations, while the remaining 8 exhibited the wild-type gene (Figure 2b). Of the 20 CFU-GM colonies, only 4 were positive (in cis) for the double mutation, while the remaining 16 were wild-type (13 colonies) or only expressed the W515L substitution (3 colonies) (Figure 2b). These data suggest a more significant heterogeneity in CFU-GM compared to BFU-E colonies and the existence of two distinct clones carrying different mutations (single or double in cis) [23].
To further analyze the genetic profile of the disease and understand its possible clinical evolution, we performed an NGS analysis on the DNA obtained from the PB of the patient, employing the same procedures described above. Sequencing data confirmed both the V501A and the W515L mutations with a VAF of 38.5% and 34.4%, respectively. The analysis also detected the KIT M541L mutation (VAF 59.7%), frequently reported in mastocytosis and chronic eosinophilic leukemia [3,24,25] (Table 4).

Discussion
In the setting of myeloproliferative diseases, knowledge about gene mutations increasingly plays a leading role. In all the newly developed prognostic models, unfavorable mutations in several genes are considered central to establishing the prognosis [26]. Gene analysis has become a widely used procedure that has made it possible to identify cases of concurrent alterations, possibly also for the same gene. In this setting, double mutations in the MPL gene are extremely uncommon in patients affected by BCR-ABL1-negative MPNs [11][12][13]. In our cohort of patients, diagnosed from 2012 to date, the frequency of MPL mutations is around 3% (44 out of 1244 total patients), and of these, only the two patients reported in this work presented a double mutation on the gene. Likewise, concurrent mutations in JAK2 and MPL are rare but can co-exist in subjects with ET or with myelofibrosis exhibiting myeloid metaplasia [27,28]. Indeed, the first case reported in this paper is the only one in our cohort.
Sanger sequencing and NGS are currently used to investigate the mutational profile of MPN patients. Sanger sequencing is the least sensitive technique and can discern double mutations only if they are expressed at or above the 20% threshold. However, SS may help discriminate between cis and trans mutations after clonal selection of single BFU-E or CFU-GM colonies. On the contrary, NGS analyzes multiple genetic regions with a sensitive limit of detection (i.e., <1%) and high accuracy, discerning nucleotide variants, small insertions and deletions, copy number variations, and fusion transcripts [29].
We report an ET patient simultaneously carrying the V501A and the W515R mutations. Next-generation sequencing analysis of her BM biopsy also detected a JAK2 V617F alteration. The lower frequency of the JAK2 substitution (39.77%) in the BM compared to MPL (100%) might explain the absence of the former alteration in the patient's PB and suggest progressive disease evolution from a pre-existing dominant MPL-mutated clone [30]. The second case concerns a PMF patient with a rare MPL V501A-W515L double mutation, likely generated in cis by a hematopoietic precursor mostly differentiating along the erythroid lineage. While the MPL W515L mutation is known to promote EPO-independent activation of the receptor [31], the pathogenic role of the V501A substitution is still unclear. Recently, Bridgford reported that the non-canonical V501A MPL mutation causes cytokine-independent growth in Ba/F3 cells [32], which could contribute to the disease's pathogenesis.
To date, the presence of numerous (ET patient) or limited (PMF patient) additional genetic alterations has not yet translated into a more aggressive disease, as both patients are currently in excellent clinical conditions while only receiving 100 mg daily acetylsalicylic acid for thromboembolic event prophylaxis, with a follow-up of more than 5 years since diagnosis. However, the employment of a new commercially available NGS panel specific for MPNs or myelodysplastic syndromes could be of great interest to explore other novel genetic and/or epigenetic mechanisms involved in the pathogenesis of the disease. Indeed, we recently extended NGS analysis in MPNs using a DNA/RNA panel including 40 genes and 29 fusion transcripts involved in myeloid malignancies. This new NGS panel is of great interest as it is able to confirm the diagnosis and discriminate genetic profiles involved in clonal evolution and disease progression of MPN/MDS.
In conclusion, NGS analyses coupled with colony-forming assays allow the characterization of the molecular landscape and clonal evolution of MPN patients with uncommon genetic mutations. A longer follow-up will be required to address the prognostic impact of this information.