With respect to driver mutations, JAK2 mutations were detected in 116 patients (65%). One hundred twelve of these patients carried the canonical JAK2 V617F substitution, whereas 4 carried mutations in JAK2 exon 12. All patients with exon 12 mutations in our cohort had PV. CALR mutations were detected in 32 patients (18%) with 18 patients carrying type I/type I-like mutations.17 MPL mutations were detected in 12 patients (7%). The prevalence of JAK2 V617F, CALR, and MPL mutations within each MPN subtype is listed in Table 1. Of 163 patients with classical MPN and MPN-unclassifiable (MPN-U), 13 patients (8%) were identified as TN, with no canonical driver mutations.
Of 107 patients with MF, 47 (44%) had an HMR profile based on harboring at least 1 mutation in ASXL1 (n = 36, 33%), EZH2 (n = 10, 9%), IDH1 (n = 3, 2.8%), IDH2 (n = 2, 1.8%), or SRSF2 (n = 11, 10%). Within each DIPSS risk category, HMR profile was detected in 2/11 (22%) patients with low risk, 15/37 (41%) patients intermediate-1 (Int-1) risk, 25/47 (53%) patients with intermediate-2 (Int-2), and 6/12 (50%) patients with high-risk disease.
The median total number of mutations per patient in the study cohort was 2. The median number of mutations was highest in post-MPN AML with 4 mutations followed by MF with 2 mutations, and 1 mutation in both PV and ET. The relative proportion of the number of mutations detected by TAR-SEQ varied across the different MPN subtypes as shown in Fig. 3. The highest proportion of patients with ≥3 mutations were those with post-MPN AML, followed by MF, PV, and ET, respectively (P < 0.0001, Fisher exact test). Conversely, ET had the highest proportion of patients with 1 mutation, followed by PV, MF, and post-MPN AML, respectively.
Diagnostic utility of TAR-SEQ: Establishing clonal hematopoiesis in TN MPN
Of the 13 TN patients with chronic phase MPN, TAR-SEQ established evidence of clonal hematopoiesis by identifying mutations in other genes in 8 (62%) patients (Table 2). These included 4 patients with MF (2 with PMF, 2 with PET-MF) and 4 with MPN-U. The 2 patients with PMF had an HMR profile due to the presence of ASXL1 mutations, whereas the 2 patients with PET-MF had no evidence of HMR. In 7 of the total 8 patients, mutations identified by TAR-SEQ were the only source of evidence of clonality, whereas 1 patient with MPN-U (ID 188) also had abnormal cytogenetics. In the 5 remaining TN patients, TAR-SEQ did not identify any mutations in the tested genes (Table 2).
Optimizing clinical decisions in HCT-eligible patients with intermediate (Int)-risk MF
In our cohort of 107 MF patients, 60 were HCT-eligible (56%) and 47 were HCT-ineligible (44%). The HCT-ineligible patients included 46 patients with age >70 years, prohibitive comorbidities and/or poor performance status, and 1 patient who declined receiving blood products due to religious beliefs. In HCT-eligible patients with DIPSS low- or high-risk disease, TAR-SEQ did not influence clinical decisions. HCT was recommended in all 6 patients with high-risk disease irrespective of their HMR status (present in 4 patients). All 11 patients with low-risk disease were not considered for HCT including 2 patients that had HMR mutations, though these 2 patients were monitored more closely. Of these 2 patients, 1 was lost to follow up and the other remains stable at last follow up.
HMR status influenced clinical decisions in HCT-eligible patients with DIPSS Int risk, specifically in relation to HCT candidacy in Int-1 risk disease and HCT timing in Int-2 risk disease. Of the 27 patients with Int-1 risk disease, 9 (33%) had evidence of HMR mutation status, and were therefore considered for HCT (individual cases summarized in Table 3A). However, HCT was not recommended in 3 of the 9 patients because age >65 years was their only risk factor. These patients were monitored more closely, and HCT was recommended on disease progression as described in patient ID 143 (Table 3A). HCT was recommended in the remaining 6 patients, 5 of whom underwent HCT whereas 1 patient declined. Notably, 4 of the 5 patients who ultimately underwent HCT rapidly acquired additional risk factors, such as a rise in peripheral blood blasts or anemia (Table 3A). In the 18 Int-1 risk patients with no HMR on TAR-SEQ, HCT was not recommended, and these patients were considered for HCT on disease progression.
Of the 19 HCT-eligible patients with Int-2 risk disease, early HCT was recommended in all patients with HMR (n = 9, 47%). By contrast, the absence of HMR in 10 Int-2 risk patients (53%) contributed toward a delayed HCT strategy (individual cases summarized in Table 3B). These patients were also assessed in transplant clinics. In 2 of these patients, HCT was recommended due to the presence of additional risk factors including refractory anemia and thrombocytopenia (ID 68) and 9% peripheral blasts (ID 59). In the remaining 8 patients, HCT was delayed and patients were either observed (n = 4) or treated with non-HCT therapies such as ruxolitinib or hydroxyurea as indicated (n = 4, Table 3B). Seven patients remain stable at last follow up, whereas 1 patient under observation progressed to DIPSS high-risk disease and subsequently underwent HCT (ID 7). The approach to utilizing TAR-SEQ in optimizing clinical decisions in HCT-eligible patients with Int-risk MF is summarized in Fig. 4.
Optimizing risk stratification in PV/ET
In our cohort, 4/26 (15%) patients with PV and 2/21 (10%) patients with ET had 3 or more mutations detected by TAR-SEQ. Furthermore, 1 JAK2 V617F positive PV patient (ID 183) was found to be positive for a TP53 mutation. However, there is no consensus on changing clinical management in PV/ET patients with adverse risk genetic profiles. Therefore, it was decided to monitor these patients more closely.
Potential therapeutic utility of TAR-SEQ
Identifying candidates for potential future clinical trials of novel molecular targeted therapies
There are a number of novel molecular targeted therapies, which are currently under investigation in hematologic malignancies. These include inhibitors of the spliceosome machinery (eg, H3B-8800), metabolic inhibitors targeting IDH1 and 2, and inhibitors of EZH2, which is implicated in epigenetic modification (Table 4). In our cohort, TAR-SEQ identified multiple MPN patients with corresponding mutations. A total of 50 patients (28%) were found to have spliceosome mutations including SF3B1 (n = 14, 8%), SRSF2 (n = 18, 10%), U2AF1 (n = 23, 13%), and ZRSR2 (n = 1, 0.6%). Of these patients, 45 were in chronic phase MPN and 5 with post-MPN AML. Seven patients (4%) were identified with mutations in IDH1 or IDH2, 5 of which were in chronic phase MPN and 2 with post-MPN AML. Finally, 13 patients (7%) with chronic phase MPN were found to have EZH2 mutations: 10 with MF, 2 with MPN-U, and 1 with ET. Some of these patients may potentially be considered as candidates for future clinical trials of the novel agents currently under clinical investigation in other hematologic malignancies (Table 4).
TAR-SEQ in the management of post-MPN AML
In the 12 patients with post-MPN AML, 4 (33%) carried mutations in TP53. Five patients (42%) could be potential candidates for future clinical trials of novel targeted therapies due to the presence of mutations in IDH1/IDH2 or in the spliceosome machinery (Table 4). Of note, 3 of the 12 patients died within 6 weeks of diagnosis, and others progressed rapidly. Clinical decisions regarding treatment with chemotherapy or hypomethylating agents were therefore already made in these patients before TAR-SEQ results were available.
Our study demonstrates the feasibility of integrating clinical grade genomic profiling using TAR-SEQ in the routine clinical workflow of MPN patients. The ability to perform TAR-SEQ upfront on initial patient referral, along with the high consent rate (98%) enabled us to accurately represent the utility of TAR-SEQ in a “real life” clinical setting. TAR-SEQ showed clinical utility in (a) diagnosis, through verifying clonal hematopoiesis in TN patients; (b) refining clinical decisions relating to HCT in Int-risk MF, and (c) identifying potential candidates for future clinical trials of novel targeted therapies.
Establishing evidence of clonal hematopoiesis through molecular assays in TN MPN has been emphasized in the latest WHO classification of myeloid neoplasms because some patients diagnosed with TN MPN may have a nonclonal disorder of hematopoiesis.2 Furthermore, there are rare conditions that can mimic the peripheral blood abnormalities and bone marrow morphology features associated with MPN, leading to misdiagnosis. We have observed 2 cases of angiosarcoma of the liver with metastases to bone in which bone marrow biopsy was reported as PMF, and subsequent investigations including liver biopsy confirmed the diagnosis. Similar cases have been reported in the literature.18,19 The presence of a clonal marker would therefore complement the morphological diagnosis of MPN. Conversely, absence of a clonal marker may heighten the suspicion for secondary etiologies or nonclonal disorders, warranting careful evaluation.
There is no consensus on the list of genes that should be screened for mutations in TN MPN. While the WHO recommends screening for the most frequently mutated genes (ie, ASXL1, EZH2, TET2, IDH1, IDH2, SRSF2, and SF3B1), given the diversity of mutations occurring in MPN, mutations that establish clonality may occur in other infrequently mutated genes. Of the 8 patients in which TAR-SEQ demonstrated evidence of clonality in our cohort, 2 lacked the most frequent accompanying mutations recommended by the WHO (Table 3). Using an NGS-based gene panel would therefore increase the probability of identifying clonal markers in TN patients. However, despite the broad coverage of commercially available TAR-SEQ panels, there still remain 4 TN patients in our cohort with no clonal markers identified. Diagnostic work up was carefully reviewed in these patients. These patients are being investigated further in a research setting with more extensive sequencing methods such as whole genome sequencing.
Current expert guidelines recommend that clinical decisions in MF should be made based on the DIPSS score, with HCT conventionally offered for patients with Int-2/high-risk disease, and selected patients with Int-1 disease.20 However, in practice the decision to select HCT versus non-HCT-based therapy is complex and involves careful review of various patient, disease, and HCT-related factors.21 The wider availability of JAKi, which can help in improving the symptom burden of the disease, has further complicated this decision, particularly the timing of HCT. Furthermore, patients with Int-1/2 risk disease are a heterogeneous population with respect to disease severity and risk of LT, warranting further risk stratification to optimize clinical decisions. NGS is an additional tool that can be integrated in clinical practice to predict disease-related risk as HMR positive Int-1 risk patients have a shorter survival and/or higher risk of LT making them potential candidates for early HCT (Table 3A). On the contrary, patients lacking HMR may have durable responses to JAKi therapy and can therefore be considered for delayed HCT. Our study showed that several patients with Int-2 risk disease, who lacked an HMR profile on TAR-SEQ had durable responses with JAKi therapy (Table 3B).
In contrast to the heterogeneous patient population with Int-risk disease, TAR-SEQ did not impact HCT decision making in patients with DIPSS high- or low-risk disease. DIPSS high-risk patients are known to have poor prognosis with a median survival of 1.5 years even in the absence of an HMR profile,5 and a shorter TTF with JAKi therapy (hazard ratio 2.79).9 Therefore, HCT was recommended for all eligible patients in this risk category irrespective of HMR status. Conversely, the immediate morbidity and mortality associated with HCT likely outweighs the additional risk conferred by the HMR profile in DIPSS low-risk patients, who are typically asymptomatic with a good quality of life.21 Thus, these patients were not considered for HCT, but patients with an HMR profile were monitored more frequently such that HCT can be considered on disease progression.
Although this study provides a framework for how NGS could be used in practice to inform clinical decisions, the impact of integrating NGS testing on improving the overall outcomes in MF remains unknown, and should be interrogated in well-designed prospective studies. We have developed a framework for integrating NGS in the management of MF for further evaluation in a multicenter study through the Canadian MPN Group (Supplementary Fig. 1, Supplemental Digital Content, http://links.lww.com/HS/A5). Recently, 2 prognostic scores that incorporate mutations in risk stratification for MF are described.22,23 Clinical integration of mutational profiles in routine clinical work flow will further facilitate the use of these refined scores.
Another advantage of using NGS-based gene panels for risk stratification of MPN over individual gene testing is that the definition of HMR is a “moving target” that will likely further change with time as more data from mutation carriers are collected and validated in different patient cohorts. In addition to TP53, an association between adverse outcomes and mutations in several genes outside the current HMR definition including CBL, CEPBA, KIT, RUNX1, SH2B3, and TET2 have been reported in retrospective studies,7,24 warranting further evaluation.
There is no clear HMR definition in PV/ET at present. A case of particular interest in our cohort was a PV patient who carried a TP53 mutation. Long-term careful follow-up of such patients will help in establishing their clinical course. Mutational profiles may also have implications in predicting thrombosis risk, which is an essential component in the management of PV/ET. A recent study showed that mutations in DNMT3A and TET2 are associated with accelerated atherosclerosis and coronary artery disease in hematologically normal individuals with clonal hematopoiesis of indeterminate potential.25 It is tempting to speculate that the risk of thrombotic complications in PV/ET patients carrying these mutations may be further increased. Of our 47 patients with PV/ET, 5 patients had mutations in DNMT3A and/or TET2. Future studies should examine whether these patients have an increased predisposition to cardiovascular thrombotic complications. If proven, this will have a major impact on screening strategies for cardiovascular disease in MPN patients.
Identifying specific genetic mutations amenable to targeted therapy is the cornerstone of personalized medicine. In a significant proportion of our cohort, TAR-SEQ detected mutations in genes that are targets for various molecular-targeted therapies currently under clinical investigation in hematologic malignancies including inhibitors of the spliceosome machinery, IDH1, IDH2, and EZH2. There are currently no targeted therapy trials in MPN, perhaps due to the rarity of these disorders, and a small pharmaceutical market. It is hoped that these data will bring attention to MPN patients for such clinical trials. Investigating novel therapies either as monotherapy or in combination with JAKi is potentially plausible in MF patients who are unlikely to have a durable response with JAKi, or after failure of JAKi therapy. In addition, treatment outcomes for post-MPN AML patients remain unsatisfactory with no improvement in survival made in the last 20 years26 further encouraging the investigation of novel agents in this poor prognosis patient population.
Finally, it is important to emphasize practical limitations encountered when using NGS that are hindering wider implementation in routine clinical practice. The current process of variant calling is labor intensive, and there is considerable variability in variant annotation between different laboratories. Thus, there is an urgent need to develop standardized criteria for variant classification.27 Furthermore, current somatic variant classification guidelines do not address finer nuances such as complex interactions between variants within gene pathways. Optimizing turnaround time for test results is also essential, especially in patients with post-MPN AML, who can progress rapidly before the results can be used to inform clinical decision making. The advent of artificial intelligence-enabled variant assessment technologies amenable to machine learning may potentially overcome some of these problems in the future.28
In conclusion, this study provides evidence on potential uses of NGS in the routine clinical setting, and provides a framework for integrating NGS in the workflow of MPN. Continuous enrollment in clinical trials or prospective registries is important for further evaluation of the impact of these technologies on improving outcomes of MPN patients.
The authors thank to the patients who contributed to this study.
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Supplemental Digital Content
Copyright © 2018 The Authors. Published by Wolters Kluwer Health Inc., on behalf of the European Hematology Association.