Clin Lymphoma Myeloma Leuk 16: S105-S113

Prognosis of primary myelofibrosis in the genomic era

Introduction

Primary myelofibrosis (PMF), the most aggressive of the classic Philadelphia chromosome-negative (Ph^{) myeloproliferative neoplasms (MPNs),}¹ is a clonal, stem-cell disorder clinically characterized by anemia, splenomegaly, extramedullary hematopoiesis, a variety of constitutional symptoms, and relatively short survival.² In a Swedish population-based study of 9,384 individuals with Ph^{MPNs diagnosed from 1973 through 2008, survival was found to have improved significantly over time; however, the improvement was less pronounced after the year 2000 and was confined to patients with polycythemia vera (PV) and essential thrombocythemia (ET).}³ Another European study examined survival trends amongst patients diagnosed with PMF between 1980 and 1995 (n=434) and between 1996 and 2007 (n=368), and found a significant improvement in median survival between the two eras (4.6 vs. 6.5 years); however, reduction in disease-specific mortality was restricted to the lower risk categories (discussed below), with no improvement in survival of intermediate-2 or high risk patients.⁴ Recently, the Janus kinase (JAK) 1/2 inhibitor, ruxolitinib, approved by the Food and Drug Administration (FDA) for the treatment of patients with intermediate or high risk patients with PMF, post-PV or post-ET MF, has demonstrated a survival benefit for these poor risk categories of patients in randomized, controlled clinical trials,⁵,6 in comparison to matched historical controls,⁷,8 as well as in a meta-analysis of pivotal, registration trials in the United States and Europe.⁹

Current prognostic classification systems in PMF

Although a number of prognostic scoring systems have been used over the years (reviewed in ref.¹⁰), robust prognostic modelling in PMF began with the publication of the International Prognostic Scoring System (IPSS) by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) in 2009.¹¹ This simple and widely used system uses 5 clinical variables - age >65 years, constitutional symptoms, hemoglobin <10 g/dl, leukocyte count >25 × 10^{/l and circulating blasts ≥1% - each assigned one point, to delineate four prognostic categories: low, intermediate-1, intermediate-2 and high. The respective median survivals in these four categories were 135, 95, 48 and 27 months in the original cohort of 1054 consecutively diagnosed PMF patients.}¹¹ In the IPSS dataset, patients without splenomegaly at diagnosis survived longer than those with splenomegaly, but the difference did not reach statistical significance, and including splenomegaly at diagnosis as a variable did not improve the prognostic model.¹¹

The IPSS risk factors were then validated at later time points, leading to the development of the Dynamic IPSS (DIPSS), which can be used at any time point in the patient's clinical course.¹² Anemia (hemoglobin <10 g/dl) was assigned 2 points in this model, with the other IPSS clinical variables receiving 1 point each. Median survivals were not reached, 14.2, 4 and 1.5 years for low, intermediate-1, intermediate-2 and high risk patients, respectively.¹² Importantly, the DIPSS has also been shown to predict progression to blast phase (BP) in PMF.¹³ Additionally, its usefulness in predicting outcomes after allogeneic hematopoietic cell transplantation (HCT) has been shown.¹⁴

Recognition that unfavorable cytogenetic abnormalities,¹⁵-18 red blood cell (RBC) transfusion dependence¹⁹ and thrombocytopenia¹⁵,17 impact prognosis in PMF led to the refinement of the DIPSS into the DIPSS-plus,²⁰ which adds these three adverse features to DIPSS risk. Thus, 1 point each is assigned to DIPSS intermediate-1 risk, unfavorable karyoptype (defined as complex karyotype, or single or two abnormalities including +8, -7/7q-, i(17q), -5/5q-, 12p-, inv(3) or 11q23 rearrangement), platelets <100 × 10^{/l, and RBC transfusion need.}²⁰ DIPSS intermediate-2 and high risk are assigned 2 and 3 points, respectively. Low (0 points), intermediate-1 (1 point), intermediate-2 (2-3 points) and high (4-6 points) risk patients had median survivals of 180, 80, 35 and 16 months, respectively.²⁰ A model that assigns 2 points to “very high risk” cytogenetics (monosomal karyotype or inv(3)/i(17q)) and 1 point each to circulating blasts ≥2% and platelets ≤50 × 10^{/l to classify patients as being at low (0 points), intermediate (1 point) or high (≥2 points) risk for leukemic transformation (LT) has been proposed by the IWG-MRT, with 3-year LT rates of 3%, 10% and 35%, respectively.}²¹

Driver mutations in the Ph^MPNs

A driver mutation is one that confers a selective advantage to a cell with self-renewal capacity, leading to the formation of a clone of mutated cells.²² Driver mutations can be founding (initiating) mutations, which give rise to the initial clone of a malignancy, or sub-clonal (cooperating) mutations, which occur in an already established clone and generate sub-clones carrying both the founding and the newly acquired mutation.²² Sub-clonal mutations are commonly associated with disease progression.²² In the case of Ph^{MPNs in particular, an important concept is that the founding “driver” mutations, while largely “driving” disease phenotype, are not necessarily the first somatic mutations leading to the development of these disorders.}²² This is elucidated in the following paragraphs, as well as in the next section.

JAK2 mutations

The observation that acquired uniparental disomy (UPD) of chromosome 9p (loss of heterozygosity (LOH) due to mitotic recombination) was a frequent stem cell defect in PV²³ set the stage for the discovery in 2005 of the activating JAK2 V617F mutation,²⁴-27 found in approximately 95% of patients with PV and 50-60% of patients with ET and PMF. This mutation in the “pseudokinase” domain of JAK2 is unique to myeloid malignancies and removes its inhibitory influence on the catalytic domain, leading to constitutive activation of the kinase.²⁸ A specific constitutional JAK2 haplotype, designated 46/1 (GGCC), confers MPN susceptibility by preferentially acquiring the V617F mutation,²⁹,30 as does a germline JAK2 single-nucleotide polymorphism (SNP), rs10974944.³¹ In addition to the well-known canonical actions of JAKs in transducing signals from membrane-bound cytokine and hematopoietic growth factor receptors, both wild type and mutant JAK2 translocate to the nucleus and phosphorylate histone H3 to regulate gene expression.³² Furthermore, mutant JAK2 phosphorylates the protein arginine methyltransferase PRMT5 with much greater affinity than wild type JAK2, leading to decreased methyltransferase activity and increased myeloproliferation.³³

Expression of JAK2 V617F in mice induces a PV-like disease with secondary myelofibrosis,³⁴-38 although experimental manipulation of the JAK2 V617F allele burden can result in ET- or PMF-like phenotypes.³⁹,40JAK2 V617F homozygous mice develop a severe hematopoietic stem cell defect, suggesting that additional lesions are needed to sustain clonal expansion.⁴¹ While homozygosity for JAK2 V617F is most common in patients with PV, the mutant allele burden in patients with PMF is often equally high.⁴² In fact, the JAK2 V617F allele burden is extremely low in hematopoietic stem cells (HSCs) from PV and ET patients at diagnosis, rising only at late stages of hematopoiesis,⁴³,44 whereas it is much higher in HSCs from patients with PMF or post-PV/ET MF.⁴⁵,46 The JAK2 V617F mutation appears to provide only a minor advantage to HSCs, such that on its own, it would cause disease with a very long latency.⁴⁷,48 Therefore, cooperation with other genetic events modifying HSC biology would greatly facilitate the development of the MPN phenotype.⁴⁹ It has been suggested that JAK2 V617F-bearing HSCs remain harmless for a long time, until genetic or environmental changes such as hematopoietic stress or aging allow clonal dominance and MPN emergence.⁴⁹ Indeed, PMF has been considered an “accelerated phase” of the classic Ph^MPNs.⁴⁹,50 Finally, there is considerable evidence to support the acquisition of JAK2 V617F as being a late event in at least some patients with Ph^MPNs,⁵¹-54 and nullizygosity for the JAK2 46/1 haplotype has been associated with shortened survival regardless of the presence or absence of the V617F mutation.⁵⁵ Taken together, these observations point to the underlying genomic complexity of Ph^{MPNs, particularly PMF, and suggest that other genetic lesions are also involved in disease pathogenesis, consistent with the two-hit theory of leukemogenesis.}⁴⁹

A low, rather than high, JAK2 V617F allele burden has been associated with inferior survival and LFS in PMF.⁵⁶,57 While some studies have linked JAK2 V617F positivity to poorer survival and a higher risk of LT in PMF,⁵⁸,59 others have not,⁶⁰ and the mutation is often lost upon progression to BP.⁶¹,62

MPL mutations

Approximately 5-10% of patients with PMF bear activating mutations in MPL, the gene encoding the thrombopoietin receptor, most commonly W515L/K affecting exon 10.⁶³-65MPL mutations in PMF also arise at the stem cell level⁶⁶ and are generally mutually exclusive of JAK2 mutations. As in the case of JAK2 V617F, acquired UPD (of chromosome 1p) has been linked to homozygosity for MPL W515L,⁶⁷,68 and the 46/1 JAK2 haplotype also predisposes to MPL exon 10 mutations.⁶⁹

CALR mutations

Recently, activating mutations in the endoplasmic reticulum (ER) chaperone protein, calreticulin (CALR) were discovered in the vast majority of patients with JAK2/MPL-unmutated ET (67-71%) and PMF (56-88%).⁷⁰,71CALR mutations were not described in PV, of which nearly all cases bear activating mutations in JAK2, either in exon 14 (V617F) or 12.⁷²-74 All CALR mutations described to date (predominantly “type 1” 52-base pair deletions in PMF and less frequently “type 2” 5-base pair insertions) appear to be mutually exclusive of JAK2 and MPL mutations, affect exon 9 and result in the generation of a protein with an abnormal carboxyl terminal with loss of the ER retention motif and impaired calcium binding.⁷⁰

Patients who lack any of the afore-mentioned founding driver mutations are said to have “triple negative” disease (up to 10%).⁷⁵,76 A key concept is the uniform activation of the JAK-STAT (signal transducer and activator of transcription) pathway in Ph^{MPNs and hence, susceptibility to therapeutic JAK inhibition.}⁷⁷ A transcriptional signature consistent with activated JAK2 signaling is seen in all Ph^{MPN patients regardless of clinical phenotype or mutational status.}⁷⁸ Indeed, the clinical benefit of ruxolitinib in patients with PMF or post-PV/ET MF in the pivotal COMFORT trials was observed regardless of JAK2 mutation status,⁷⁹,80 and the drug has specifically been shown to be an effective treatment for patients with CALR-mutated PMF.⁸¹

Two large studies have examined the clinical features and outcomes of molecularly annotated patients with PMF.⁷⁵,76 In the Italian study in 617 subjects, median survival was 17.7 years in CALR-mutant, 9.2 years in JAK2-mutant, 9.1 years in MPL-mutant, and 3.2 years in triple-negative patients.⁷⁵ Patients with CALR mutation were younger, had higher platelet counts, and a lower risk of developing anemia, thrombocytopenia, and marked leukocytosis compared with other subtypes.⁷⁵,76 Both studies demonstrated a favorable effect of CALR mutations on survival and LFS and documented the poor survival and LFS of “triple negative” patients. Furthermore, the impact of genetic lesions on survival was independent of the current prognostic scoring systems.⁷⁵,76 However, in contrast to the Mayo Clinic experience,⁸² the Italian investigators did not find major differences in phenotype or prognostic impact between type 1 and type 2 CALR mutations.⁷⁵ The favorable effect of CALR mutations on survival and the poor survival of triple negative patients as compared to JAK2/MPL-mutated patients has also been shown in the post-allogeneic HCT setting in a German study (n=133, 97 PMF, 36 post-PV/ET MF) on univariate analysis.⁸³ However, on multivariate analysis, only older age and female donor sex remained independent predictors of shorter survival, with CALR mutation status showing a trend for better survival.⁸³

The Italian invesigators also developed as a proof-of-concept a “clinical molecular prognostic model” to see if information on JAK2, MPL and CALR mutation status improves IPSS-based risk stratification.⁷⁵ They assigned 1 point each for presence of constitutional symptoms, circulating blasts ≥1%, hemoglobin <10 g/dl and presence of a JAK2 mutation, and 2 points each for presence of a MPL mutation, triple negativity, leukocytes >25 × 10^{/l, and age >65 years.}⁷⁵ The resulting risk categories were very low (score 0), low (score 1), intermediate (score 2-3), high (score 4-5), and very high (score ≥6). The survival curves generated in this manner were significantly different, and the model performed better than the IPSS using established statistical criteria.⁷⁵

Mutations in negative regulators of JAK2

A small proportion of patients with Ph^{MPNs bear mutations in}CBL and LNK, encoding two negative regulators of JAK2 signaling. Loss-of-function mutations in the inhibitory adaptor protein LNK were first described in two patients with JAK2 V617F-negative MPNs as a novel mechanism of JAK-STAT activation.⁸⁴LNK mutations are not mutually exclusive, however, of JAK2 V617F and may be more common in BP PMF.⁸⁵ Gain-of-function mutations in CBL are strongly associated with acquired UPD of chromosome 11q, ultimately leading to loss of its tumor suppressor function through loss of the wild type alleles.⁸⁶,87CBL mutations have been reported to occur in 6% of PMF patients,⁸⁸ and may play a larger role during LT.⁴⁹,89

JAK2 mutations

MPL mutations

CALR mutations

Mutations in negative regulators of JAK2

Mutations in epigenetic regulators, splicing genes and genes involved in leukemic transformation

Mutations in a host of other genes are commonly encountered in patients with Ph^{MPNs, particularly PMF. Most of these affect epigenetic regulators and spliceosome components, as in the myelodysplastic syndromes (MDS).}⁹⁰ The major ones, especially those that have been shown to impact prognosis in PMF, are discussed below. Less frequently occurring mutations (3-8% of patients) in other genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG, NRAS) have also been described.⁹¹

TET2 mutations

The Ten-Eleven-Translocation (TET) enzymes hydroxylate 5-methylcytosine (mC) to produce 5-hydroxymethylcytosine (5hmC).⁹²-94 Although named after TET1, located on chromosome 10, TET2 is located on chromosome 4q24, and UPD and LOH of 4q24 have been associated with TET2 mutations in MDS⁹⁵ and MDS/MPN overlap syndromes.⁹⁶ In animal models, loss of TET2 leads to progressive enlargement of the HSC compartment and eventual myeloproliferation, including splenomegaly, monocytosis, and extramedullary hematopoiesis.⁹⁷ 5hmC is believed to represent a step towards cytosine demethylation,⁴⁹ which is consistent with the observed clinical benefit from hypomethylating agents in TET2-mutated MDS.⁹⁸TET2 mutations occur across the spectrum of myeloid malignancies, and in the original report, were found in 12% of patients with Ph^MPNs.⁹⁹ Importantly, TET2 mutations can precede the acquisition of JAK2 V617F in Ph^MPNs,⁹⁹ or occur as a late event during progression.¹⁰⁰ It was recently demonstrated that the order in which TET2 mutations and JAK2 V617F are acquired influences clinical characteristics, response to ruxolitinib in vitro, stem and progenitor cell biology and clonal evolution in the Ph^MPNs.¹⁰¹TET2 mutations increase in frequency with age, even in healthy individuals, and are associated with clonal hematopoiesis and an increased risk of future hematologic cancers.¹⁰²-104 In one study, no effect on survival or LT was found among 60 PMF patients, 17% of whom had TET2 mutations,¹⁰⁵, but other investigators have found TET2 mutations to be associated with LT⁸⁹,106 and poor outcomes.¹⁰⁷

IDH1/2 mutations

Mutations in the genes encoding the isocitrate dehydrogenase 1 and 2 (IDH1/2) enzymes of the Krebs' cycle result in the production of the abnormal metabolite 2-hydroxyglutarate (2HG) instead of the normal α-ketoglutarate (αKG) from isocitrate.⁴⁹,108 In IDH1/2-mutant acute myeloid leukemia (AML), 2HG inhibits the function of TET2, an αKG-dependent enzyme, leading to a hypermethylation phenotype and impaired hematopoietic differentiation.¹⁰⁹ Thus, TET2 loss-of-function mutations are associated with similar epigenetic defects as IDH1/2 mutants, and the two are mutually exclusive in AML.¹⁰⁹IDH1/2 mutations have been implicated in the progression of chronic phase (CP) Ph^{MPNs to AML.}¹⁰⁶,110,111 In an analysis of IDH1/2 mutations among 200 patients with CP (n=166) or BP (n=34) MPNs, mutational frequencies were approximately 21% (7 of 34) for BP MPNs and approximately 4% (3 of 77) for PMF. Only 1 of 12 patients with paired samples available had IDH mutations both in CP and BP.¹¹² In a much larger, multi-institutional study of 1473 patients with Ph^{MPNs, the}IDH mutation frequencies were virtually identical: 4.2% in PMF and 21.6% in BP MPNs.¹¹³IDH-mutated patients were more likely to be nullizygous for the JAK2 46/1 haplotype, especially in PMF. In CP PMF, JAK2 46/1 haplotype nullizygosity, but not IDH mutational status had an adverse effect on survival.¹¹³ In contrast, in both BP PMF and BP MPN, the presence of an IDH mutation predicted worse survival.¹¹³ However, in a follow-up study in 301 consecutive patients with CP PMF, IDH mutations predicted for worse survival and LFS, particularly in JAK2 V617F-positive patients.¹¹¹

EZH2 mutations

Enhancer of Zeste homolog 2 (EZH2) is a Polycomb group protein that interacts with DNA methyltransferases (DNMTs) within the context of Polycomb repressive complexes 2 and 3 (PRC2/3) and controls the binding of DNMTs to the promoters of several genes repressed by methylation, serving as a recruitment platform for DNMTs.¹¹⁴EZH2 encodes the catalytic subunit of PRC2, a highly conserved histone H3 lysine 27 (H3K27) methyltransferase that mediates repression of gene transcription.¹¹⁵EZH2 acts as a tumor suppressor gene in myeloid malignancies,¹¹⁶ and homozygous inactivating mutations in EZH2 were first described in individuals with acquired UPD of chromosome 7q, abnormalities of which are common in myeloid neoplasms.¹¹⁷ In the original report, EZH2 mutations were found in 13% of patients with myelofibrosis.¹¹⁷ In a subsequent study of 370 subjects with PMF and 148 with post-PV/ET MF, EZH2 mutations were detected in 5.9% of PMF, 1.2% of post-PV MF, and 9.4% of post-ET MF patients.¹¹⁸EZH2-mutated patients had significantly poorer survival and LFS; on multivariate analysis, inferior survival was predicted only by IPSS high risk, low JAK2 V617F allele burden and mutated EZH2.¹¹⁸ In another study, EZH2 mutations were detected in 3 of 46 (7%) cases of PMF from one institution and none of 25 from another, as well as in no cases of post-PV/ET MF (n=22) or BP MPN (n=11); no association with clinical outcome was observed in this study.¹¹⁹

ASXL1 mutations

Additional sex combs like 1 (ASXL1) is a PRC gene required for long-term repression of homeobox genes.¹²⁰ It is part of a novel complex (Polycomb repressive deubiquitinase, PR-DUB) that removes monoubiquitin from histone H2A in nucleosomes, an action opposite to that of PRC1 and the related dRing-associated factors (dRAF) complex, thus helping maintain a dynamic balance between H2A ubiquitination and deubiquitination.¹²¹ In a small study (n=64), heterozygous ASXL1 mutations were found in one out of 35 cases of ET, 3 out of 10 cases of PMF (one in accelerated phase) and one out of 5 cases of BP MPNs (a case of post-ET AML).¹²² In a study of 63 patients with AML arising from a pre-existing Ph^MPN,ASXL1 mutations were found in 19.3%, and were almost always detected in paired samples from individual patients, suggesting that they were not acquired at LT.¹⁰⁶ Like TET2 mutations, ASXL1 mutations can precede the acquisition of JAK2 V617F.¹⁰⁶ In a larger, multi-institutional cohort, the frequency of ASXL1 mutations was found to be 12-13% in PMF and 23% in post-PV/ET MF.¹¹⁹ While an adverse impact on survival was suggested on univariate analysis, this was lost upon multivariate analysis that included DIPSS risk.¹¹⁹ However, a CALR/ASXL1 mutations-based prognostic model was found to be DIPSS-plus independent in a large international study (n=570) of PMF patients.¹²³ Survival was the longest in CALR^ASXL1^- (median 10.4 years) and shortest in CALR^ASXL1⁺ patients (median 2.3 years). CALR^ASXL1⁺ and CALR^ASXL1^- patients had similar survival and were grouped together in an intermediate-risk category (median survival 5.8 years).¹²³

Spliceosome mutations

Somatic mutations in spliceosomal genes, e.g., SF3B1, U2AF1, SRSF2, ZRSR2, etc., resulting in altered pre-mRNA splicing patterns, are ubiquitous across myeloid and lymphoid leukemias, and represent a recently discovered leukemogenic pathway.¹²⁴ In the original publication, spliceosome pathway mutations were reported to occur in 9.4% of patients with Ph- MPNs.¹²⁵ In a subsequent study in PMF, monoallelic mutations in SRSF2 were found in 32 of 187 (17%) patients, and correlated with shortened survival and LFS.¹²⁶ There was significant clustering with IDH mutations, and the adverse impact on survival was independent of both DIPSS-plus and IDH mutations.¹²⁶ In contrast, SF3B1 mutations, common in MDS with ringed sideroblasts,¹²⁷ MDS/MPN overlap syndromes¹²⁸ and chronic lymphocytic leukemia,¹²⁹,130 were found in only 10 of 155 (6.5%) of patients with PMF and did not influence survival.¹³¹

TP53 mutations

Mutations in TP53 are strongly associated with progression of Ph^{MPNs to AML.}⁸⁹,107,132-134 In one study,¹³² somatic mutations in TP53 were found in 27.3% of Ph^{MPN patients in whom LT had occurred, and 18.18% of patients had amplifications of chromosome 1q, which harbors}MDM4, encoding a negative regulator of p53 frequently overexpressed in cancer.¹³⁵TP53 mutations can be present in the heterozygous state for prolonged periods during the chronic phase of MPNs without clonal expansion; however, loss of the wild type allele by chromosomal deletion or UPD leads to rapid expansion of the hemi- or homozygous mutant TP53 clone, ultimately causing LT.¹⁰⁷ The low allele burden of mutant TP53 in the chronic phase of MPNs suggests the need for sensitive methods such as next-generation sequencing (NGS) for reliable detection, as this is clearly a high-risk group of patients.¹⁰⁷

DNMT3A mutations

Loss-of-function mutations in DNA methyltransferase 3A (DNMT3A) are common in AML¹³⁶ and are associated with poor outcomes in both AML and MDS.¹³⁶,137 In a study of 80 patients with CP MPNs (30 PV, 30 ET and 20 MF, including PMF and post-PV/ET MF) and 35 with BP MPNs, 12 DNMT3A sequence variants were identified (10% overall; BP MPNs, 17%; MF, 15%; PV, 7%; ET, 3%), of which five could be proved to be of somatic origin.¹³⁸ In a cohort of 46 patients with PMF, 22 with post-PV/ET MF, 11 with BP MPNs and 15 with chronic myelomonocytic leukemia (CMML), DNMT3A mutations were uncovered in only 3 (7%) PMF patients and in none of the others.¹³⁹ No correlation with outcomes or presenting features was identified.¹³⁹

RUNX1 mutations

Runt-related transcription factor 1 (RUNX1) encodes a heteromeric transcription factor with a major role in hematopoiesis⁴⁹ that is frequently mutated in AML¹⁴⁰ and MDS,¹⁴¹ where it has been associated with adverse outcomes. RUNX1 has been implicated in LT of Ph^MPNs.⁸⁹,133,142 Inactivating mutations in RUNX1 cause differentiation arrest and both point mutations and chromosomal aberrations at the RUNX1 locus are found in post-MPN AML samples.¹³³

IKZF1 deletions

The Ikaros family zinc finger 1 (IKZF1) gene located on chromosome 7p encodes for the Ikaros transcription factors, which are important regulators of lymphoid differentiation.¹⁴³IKZF1 gene deletions are implicated in the pathogenesis of the lymphoid BP of chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia.¹⁴⁴IKZF1 deletions have been reported to be strongly associated with the development of post-MPN AML, deletion 7p and a complex karyotype.¹⁴⁵ Hemizygous loss of IKZF1 (including monosomy 7) was detected in 21% of post-MPN AML and 0.2% of non-leukemic MPN patients.¹⁴⁵

Mutations and leukemic transformation in the Ph^MPNs

Given that a JAK2 V617F-positive MPN can evolve either into a JAK2 V617F-positive or wild type AML, two routes for LT have been proposed.⁸⁹ In the former situation, evolution to JAK2-mutant AML is preceded by the acquisition of genetic changes that give rise to the phenotype of accelerated-phase disease, manifested clinically as PMF⁴⁹,50 or myelofibrotic transformation of ET/PV.⁸⁹ Mutant JAK2-induced inhibition of Bcl-xL deamidation,¹⁴⁶ reactive oxygen species generation¹⁴⁷ and stimulation of homologous recombination¹⁴⁸ could contribute to genetic instability and the aberrant survival of DNA-damaged cells in this setting. However, the observation that CP MPN patients do not accumulate chromosomal aberrations¹³³ and somatic mutations¹⁰⁷ over time argues against the concept that JAK2 V617F induces genomic instability. In the latter situation, the chronic and leukemic phases of the disease could either be clonally related, having arisen from a shared pre-JAK2 V617F clone, or be clonally unrelated, reflecting transformation of independent stem cells.⁸⁹ As discussed above, TET2 and ASXL1 mutations can be acquired pre-JAK2 V617F,⁹⁹,106 as can deletion 20q, which is likely not a predisposing factor for acquisition of JAK2 V617F.¹⁴⁹

TET2 mutations

IDH1/2 mutations

EZH2 mutations

ASXL1 mutations

Spliceosome mutations

TP53 mutations

DNMT3A mutations

RUNX1 mutations

IKZF1 deletions

Mutations and leukemic transformation in the Ph^MPNs

Identification of a high molecular risk signature in PMF, importance of number of mutations and development of new prognostic models

A “high molecular risk” (HMR) signature was recently developed for patients with PMF in a multi-national study of 879 patients.¹⁵⁰ Genes analyzed included ASXL1, SRSF2, EZH2, TET2, DNMT3A, CBL, IDH1, IDH2, MPL and JAK2. In both the learning (n=483) and validation (n=396) cohorts, mutations in ASXL1, SRSF2 and EZH2 predicted shortened survival, but only ASXL1 mutations remained significant in the context of IPSS and DIPSS-plus, respectively.¹⁵⁰ LFS was adversely affected by mutations in IDH1 and SRSF2 in both cohorts and, additionally, by IDH2 and ASXL1 mutations in the learning cohort.¹⁵⁰ Based on these findings, PMF patients with mutations in any of these five genes were identified as belonging to a “HMR” category.

Conventional karyotyping using classic metaphase cytogenetics identifies chromosomal abnormalities in around 30% of patients with Ph^{MPNs, and SNP arrays do so in over half of patients.}¹³³ In contrast, at least one somatic mutation can be found in 90% of MPN patients.¹⁰⁷ It was recently shown that one of the strongest predictors of outcome is the number of somatic mutations in addition to JAK2, CALR or MPL.¹⁰⁷ Interestingly, in this study, the group of patients carrying either no detectable somatic mutation or a mutation in JAK2, CALR or MPL only had a particularly favorable prognosis, with no cases of LT.¹⁰⁷ While this may appear contradictory to the known poor LFS of triple negative patients, the discrepancy is likely explained by the fact that the mutation profiles of 105 genes were analyzed in the above study using NGS and allele-specific polymerase chain reaction,¹⁰⁷ unlike in studies that examined the clinical correlates of mutations in JAK2, MPL, or CALR versus triple negative status.⁷⁵,76,151 Indeed, an international study of 797 patients with PMF has shown that the number of mutated genes (from amongst the five comprising the HMR category) is predictive of both survival and LFS.¹⁵² Median survival was 2.6 years for patients with ≥2 mutations, 7 years for patients with one mutation, and 12.3 years for those with no mutations. The prognostic significance of number of mutations was independent of both IPSS and DIPSS-plus.¹⁵² ≥2 mutations were also associated with shortened LFS. However, CALR mutations favorably impacted survival, irrespective of both number of prognostically detrimental mutations and DIPSS-plus.¹⁵² More recently, targeted NGS of a panel of 28 genes recurrently mutated in myeloid malignancies was performed on samples obtained at study entry from 95 patients with MF who received ruxolitinib on a phase 1/2 study.¹⁵³ Mutations in 15 of these genes were found in all but 2 patients. Patients with ≥3 mutations of any type had significantly lower odds of a spleen response (nine-fold lower odds than those with ≤2 mutations) and a shorter time to treatment discontinuation (TTD).¹⁵³ In multivariate analysis, only the presence of ≥2 mutations and spleen response were associated with shorter TTD. Patients with ≥1 mutation(s) in ASXL1, EZH2, IDH1 or IDH2 (SRSF2 mutations were not tested for in this study) had shorter survival and TTD, and were significantly less likely to have a spleen response to ruxolitinib.¹⁵³ Of note, spleen response in this group of patients had previously been shown to correlate with improved survival.⁷ Reductions in spleen size with ruxolitinib treatment also correlated with longer survival in a pooled analysis of survival data from the pivotal COMFORT-I and –II trials.⁹ Collectively, these observations suggest that multi-gene profiling to identify prognostically detrimental mutations should be incorporated into the routine diagnostic workup of patients with MF and integrated into prognostic models for both outcome prediction and treatment planning.

Accordingly, a new mutation-enhanced IPSS (MIPSS) for PMF was recently presented.¹⁵⁴ Upon multivariable analysis of the learning cohort (n=588), the following factors remained significant for survival: age >60 years, constitutional symptoms, hemoglobin <10 g/dl, platelets <200 × 10^{/l, triple negativity,}JAK2/MPL mutation, ASXL1 mutation and SRSF2 mutation.¹⁵⁴ These adverse factors were weighted 1.5, 0.5, 0.5, 1.0, 1.5, 0.5, 0.5 and 0.5, respectively. Four risk groups with non-overlapping survival curves could thus be delineated: low (score 0-0.5), intermediate-1 (score 1-1.5), intermediate-2 (score 2-3.5) and high (score ≥4). MIPSS performed better than IPSS in predicting survival, and its performance was validated in an independent cohort of 398 patients with PMF.¹⁵⁴ High and intermediate-1/2 risk MIPSS also predicted LFS in both cohorts.¹⁵⁴ However, karyotype retained MIPSS-independent prognostic value for both survival and LFS in the validation cohort.¹⁵⁴ Because of the latter observation, a prognostic model based only on age and genomic information, the Genetics-based Prognostic Scoring System (GPSS) was developed.¹⁵⁵ Four cytogenetic risk categories were defined: very high (monosomal karyotype,¹⁵⁶ inv(3), i(17q), -7/7q-, 11q or 12p abnormalities), high (complex non-monosomal, two abnormalities not included in very high risk category, 5q-, +8, other autosomal trisomies except +9, and other sole abnormalities not included in other risk categories), intermediate (sole abnormalities of 20q-, 1q+ or any other sole translocation, and -Y or other sex chromosome abnormality) and low (normal or sole abnormalities of 13q- or +9).¹⁵⁵ Age-adjusted multivariable analysis identified the following factors as independent predictors of shortened survival in the training set of 964 patients with PMF: very high risk karyotype (3 points), high risk karyotype (1 point), triple-negative (2 points), JAK2 mutation (2 points), MPL mutation (2 points), type 2/type 2-like CALR mutation (2 points), ASXL1 mutation (1 point) and SRSF2 mutation (1 point).¹⁵⁵ Age >60 years received 2 points. Four risk categories could be demarcated in this way: low (0 points), intermediate-1 (1-2 points), intermediate-2 (3-4 points) and high (≥5 points), with corresponding median survival times of >17, 9, 5 and 2.2 years, respectively.¹⁵⁵ High-risk patients also had a higher risk of LT. The ability of the GPSS to effectively discriminate between higher (high/intermediate-2) and lower (low/intermediate-1) risk categories in terms of both survival and LFS was validated in an independent cohort of 183 patients with PMF.¹⁵⁵

Conclusion

Prognostication of PMF has traditionally relied on clinical variables, and there is no doubt that the IPSS¹¹ and DIPSS¹² are useful in classifying patients into different risk categories at diagnosis and at later time points, respectively. Although more cumbersome to use, the DIPSS-plus²⁰ added significant value to the DIPSS by incorporating cytogenetic risk. Indeed, certain karyotypic features, e.g., monosomal¹⁵⁶ or complex karyotype,¹⁶ chromosome 17 aberrations,¹⁶,17 particularly i(17q) and inv(3)¹⁸ confer an extremely poor prognosis. However, karyotypic abnormalities are not detected in the majority of patients. The explosion of knowledge in recent years of the molecular underpinnings of the disease as a result of the wide availability of techniques like NGS has uncovered a wealth of prognostically useful information, the incorporation of which into prognostic models for routine use in the clinic is just beginning. One potentially problematic issue is that all major studies of prognostic modelling in PMF to date have excluded patients with early/prefibrotic PMF, and indolent and difficult-to-diagnose entity¹⁵⁷,158 which, nevertheless, is included under PMF in the World Health Organization classification.¹⁵⁹ Another point worth noting is that there may be important biologic differences between PMF and post-PV/ET MF, such that the current practice of using the risk stratification systems developed for PMF for these patients may not be optimal.¹⁶⁰ As prognostic models in PMF continue to evolve in the genomic era, hopefully, large studies will be conducted to also address these issues.

Prithviraj Bose, Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX, USA;

Corresponding author: Prithviraj Bose, MD, Assistant Professor, Dept of Leukemia, 1400 Holcombe Blvd, FC4.3062, Unit 428, Houston, TX 77030, Phone: 713-792-7747, Fax: 713-792-7305, gro.nosrednadm@esobp

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Abstract

Currently, prognostication in primary myelofibrosis (PMF) relies on the International Prognostic Scoring System (IPSS), dynamic IPSS (DIPSS) and DIPSS-plus, which incorporate age, blood counts, constitutional symptoms, circulating blasts, red cell transfusion need and karyotype. Although the JAK2 V617F mutation was discovered a decade ago and MPL mutations shortly thereafter, it was the recent discovery of CALR mutations in the vast majority of JAK2/MPL-unmutated patients and recognition of the powerful impact of CALR mutations and “triple negative” (JAK2/MPL/CALR-negative) status on outcome that set the stage for revision of traditional prognostic models to include molecular information. Additionally, the advent of next-generation sequencing has identified a host of previously unrecognized somatic mutations across hematologic malignancies. Like in the myelodysplastic syndromes, the majority of common and prognostically informative mutations in PMF affect epigenetic regulation and mRNA splicing. Thus, a need has arisen to incorporate mutational information on genes such as ASXL1 and SRSF2 into risk stratification systems. Mutations in yet other genes appear to be important players in leukemic transformation, and new insights into disease pathogenesis are emerging. Finally, the number of prognostically detrimental mutations may affect both survival and response to ruxolitinib, which has significant implications for clinical decision making. In this review, we briefly summarize the prognostic models in use today and discuss in detail the somatic mutations commonly encountered in patients with PMF, along with their prognostic implications and role in leukemic transformation. Emerging prognostic models that incorporate new molecular information into existing systems or exclude clinical variables are also presented.

Keywords: prognosis, primary myelofibrosis, mutations, survival, leukemic transformation

Abstract

Footnotes

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Footnotes

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Prognosis of primary myelofibrosis in the genomic era

Introduction

Current prognostic classification systems in PMF

Driver mutations in the Ph MPNs

JAK2 mutations

MPL mutations

CALR mutations

Mutations in negative regulators of JAK2

JAK2 mutations

MPL mutations

CALR mutations

Mutations in negative regulators of JAK2

Mutations in epigenetic regulators, splicing genes and genes involved in leukemic transformation

TET2 mutations

IDH1/2 mutations

EZH2 mutations

ASXL1 mutations

Spliceosome mutations

TP53 mutations

DNMT3A mutations

RUNX1 mutations

IKZF1 deletions

Mutations and leukemic transformation in the Ph MPNs

TET2 mutations

IDH1/2 mutations

EZH2 mutations

ASXL1 mutations

Spliceosome mutations

TP53 mutations

DNMT3A mutations

RUNX1 mutations

IKZF1 deletions

Mutations and leukemic transformation in the Ph MPNs

Identification of a high molecular risk signature in PMF, importance of number of mutations and development of new prognostic models

Conclusion

Abstract

Footnotes

References

Driver mutations in the Ph^MPNs

Mutations and leukemic transformation in the Ph^MPNs

Mutations and leukemic transformation in the Ph^MPNs