Advances in molecular evaluation of myeloproliferative neoplasms

Myeloproliferative neoplasms (MPN) are characterized by clonal proliferation of hematopoietic stem cells and encompass several distinct entities. The advent of next generation sequencing (NGS) has greatly advanced the understanding of the molecular pathogenesis of MPN. Molecular aberrations that initiate and drive the development and progression of most MPN have been well-defined, which involve gene fusions or mutations that activate protein tyrosine kinase pathways and lead to uncontrolled cell proliferation. These molecular aberrations have been used for the diagnosis, classification, detection of minimal/measurable residual disease (MRD), and target therapy. The latest 5th edition of the World Health Organization (WHO) classification of myeloid neoplasms has classified MPN into eight subtypes.1 We review recent advances in molecular genetic aberrations of MPN with a focus on MPN associated with gene rearrangements or mutations that activate tyrosine kinase pathways (Table 1).

Chronic myeloid leukemia (CML) is molecular defined and driven by the presence of BCR::ABL1 as a result of t(9;22)(q34;q11.2), resulting in the Philadelphia chromosome (Ph). With the refinement of tyrosine kinase inhibitor (TKI) therapy and close disease monitoring, the incidence of progression to advanced phase disease has decreased. However, blast transformation occurs in a small group of patients, as a consequence of persistent BCR::ABL1 tyrosine kinase activity which induces genetic instability, leading to clonal evolution with acquisition of additional chromosomal abnormalities (ACAs) and aberrations in genetic and epigenetic pathways through gene mutations. The 5th edition of WHO classification omitted the terminology of accelerated phase (AP) in favour of an emphasis on high risk features associated with disease progression and resistance to TKI.1 The number of genetic alterations increases during the transition from chronic phase (CP) to blast phase (BP). Therefore, the genetic alterations, rather than clinical variables, contribute to a better prediction of prognosis for patients in BP.2 The International Consensus Classification (ICC) maintained but simplified the diagnostic criteria for AP, defined by 10-19% blasts in bone marrow (BM) or peripheral blood (PB), PB basophilia >20%, or the detection of ACAs.3 Both the 5th edition of WHO and ICC emphasized the presence of bona fide lymphoblasts, regardless of the percentage, in defining impending blast phase.1,3

The goal of CML treatment is to achieve a sustained early and deep molecular response and treatment-free remission and normalization of survival. The criteria for cytogenetic and molecular responses are listed in Table 2. Quantitative real-time RT-PCR is now the “gold standard” for CML monitoring. It can be done on PB or BM and is recommended at initial diagnosis and every 3 months thereafter, ideally using an international reporting scale. With a sensitivity of 0.01%-0.001%, it allows for MRD monitoring after achievement of complete cytogenetic remission (CCyR) and stem cell transplantation to assess eligibility for treatment discontinuation.4 Cytogenetic response has been regarded as a major prognostic factor. Early achievement of CCyR within 12 months decreases the chance of disease progression.5 Although less sensitive (sensitivity 5%), karyotypic analysis is the only method that can detect ACAs, and is recommended at initial diagnosis, every 6 months till achievement of CCyR, once a year thereafter, and at time of treatment resistance and disease progression. FISH can detect cryptic or atypical translocations, with a sensitivity of 0.5% and a quick turn-around time. It can be done on interphase cells using PB, and is therefore particularly useful when BM metaphase is not available.

The presence of ACAs at baseline or during TKI therapy has been associated with increased risk of TKI failure and disease progression.6, 7, 8, 9 The European LeukemiaNet (ELN) recommendations identified +8, a second copy of Ph, i(17q), +19, -7/7q-, 11q23 or 3q26.2 rearrangements, and complex karyotypes as high-risk ACAs.4 Wang et al. studied the differential prognostic impact of individual ACAs and stratified the common ACAs into two groups: group 1 included +8, -Y, and an extra copy of Ph with a relatively good prognosis; and group 2 included i(17), -7/del7q, and 3q26.2 rearrangements with a relatively poor prognosis.6 Occasionally, cases of CML acquire recurrent translocations or inversions that are typically seen in de novo acute myeloid leukemia (AML), such as t(3;21)(q26;q22), t(8;21)(q22;q22), t(15;17)()(q24.1;q21.2), and inv(16)(p13q22), at time of blast transformation.10, 11, 12, 13 Although these cases resemble de novo AML with the same translocations or inversions morphologically and immunophenotypically, the patients usually have a more aggressive clinical course and require more intensive intervention.10, 11, 12, 13 Furthermore, 10-15% of CML patients have ACAs in Ph-negative clones.14 One study found that, except –Y, clonal ACAs in Ph-negative cells were associated with decreased survival.14 Another study reported that CML patients with -7/del7q in Ph-negative cells more frequently showed myelodysplasia and had a lower cumulative incidence of deep molecular response and shorter survival.15

ABL1 kinase mutation represents the most common mechanism of resistance to TKIs, occurring in approximately half of the CML cases with TKI resistance. As different mutations vary in the resistance profile, not only the presence but also the type of point mutations may affect treatment decision. Sanger sequencing, though with a low sensitivity of 20%, is still the mainstay in the initial assessment of ABL1 mutation. More sensitive assays, such as mutation-specific PCR (sensitivity 0.01-0.001%), may be suitable for follow-up on a previously identified mutation. Targeted NGS (sensitivity 1%) can simultaneously detect mutations in ABL1 as well as other genes. For treatment naïve patients, ABL1 mutation analysis is indicated when they present with advanced phase disease. For patients on TKI therapy, ABL1 mutation analysis is indicated when they show inadequate initial responses, loss of previously attained responses, or evidence of disease progression. Detection of mutations with low variant allele frequencies (VAF) would allow early change of therapy and reduce risk of disease progression.

Genome-wide mutation analysis has identified somatic mutations in cancer-related genes other than ABL1 at diagnosis and disease progression, particularly in genes involved in chromatin modification and DNA methylation. The impact of these mutations varies depending on when they are acquired, the type and load of mutations, and the dynamics and patterns of their appearance and disappearance. Myeloid leukemia-associated mutations occur in 30-50% of newly diagnosed CML patients.16, 17, 18, 19 ASXL1 has been reported as the most frequently mutated gene at baseline CP, seen in about 10% of the cases.16, 17, 18,20, 21, 22, 23 Other recurrent mutations that have been frequently reported in CP affect DNMT3A, IKZF1 (mutations and deletions), RUNX1, TET2, and TP53.16, 17, 18, 19,21,24 Data regarding prognostic significance of these mutations are inconsistent. Some earlier studies showed no association between mutations and response to TKI or disease phase.25 However, most of the recent studies have shown that mutations affecting epigenetic modifier genes in CP-CML adversely impact response to TKI and predispose to disease progression.3,17,21, 22, 23,26,27 Schonfeld et al reported that patients carrying ASXL1 mutation at diagnosis had a less favourable molecular response to nilotinib and were more commonly found in the high-risk group.23 Bidikian et al showed CP-CML with ASXL1 mutation was associated with inferior event-free survival and failure-free survival.22 More recently, Adnan Awad et al. showed that ASXL1 mutation may be associated with disease recurrence upon discontinuation of TKI.28

It has been reported that additional gene mutations other than ABL1 have been detected in 80-90% of CML patients in BP.3,29RUNX1 is the most frequently mutated gene in BP, with a reported frequency of 20-30%, followed by ASXL1 and IKZF1 (mutations and deletions).3,22,27,29,30 Other recurrent mutations in BP include BCOR/BCORL1, CBL, CDKN2A/B, DNMT3A, EZH2, GATA2, IDH1, IDH2, JAK2, KRAS, NRAS, PHF6, PTPN11, SETBP1, TET2, TP53, and WT1.3,19,29 Mutations that frequently occur in AML, such as FLT3 and NPM1, are rare in BP-CML. It has also been shown that different BP phenotypes exhibit distinct mutation profiles, with CDKN2A/B and IKZF2 deletions more commonly seen in lymphoid BP, ASXL1 and TP53 mutations more enriched in myeloid BP, and others such as RUNX1 mutations occur in both BP.3,29,31RUNX1 mutation has been identified as an independent predictor of worse prognosis in BP-CML.22,29 Other mutations that have been associated with inferior responses to TKI and shorter survival include ASXL1, IKZF1, TP53 and WT1.3,29 Patients with concurrent TP53 mutation and del(17p) or i(17q), with presumable loss of biallelic TP53, have an especially grim clinical outcome.3

Currently there is no well-established guideline on how to use the emerging genetic and epigenetic data for risk assessment and treatment decision. It is notable that some of these genes are common examples of clonal hematopoiesis of indeterminate potential (CHIP) genes and may exist even before the acquisition of BCR::ABL1.32, thus should be interpreted with caution. Moreover, similar to ACAs, mutations of myeloid-related genes have also been detected in Ph-negative clones. For example, Schmidt et al detected gene mutations in Ph-negative clones in about 40% of CML patients receiving TKI, including DNMT3A, EZH2, RUNX1, TET2, TP53, U2AF1 and ZRSR2.16 Their roles in CML clonal evolution remain to be elucidated. The National Comprehensive Cancer Network (NCCN) recommends that a myeloid mutation panel be considered for CML patients with advanced phase disease to identify BCR::ABL1-independent mutations.33 Well-designed NGS panels to search for new mutations, deletions and/or fusions, as well as studies to explore the roles of these genetic aberrations in TKI resistance and disease progression will improve risk stratification, guide treatment decision, and aid in the development of novel therapeutic targets.

Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are the most common subtypes of Ph-negative MPNs. The overlapping clinicopathologic features reflect the presence of a shared genetic origin that activates the JAK/STAT pathway early in the disease course, the three cardinal driver mutations, JAK2, CALR, and MPL.34, 35, 36, 37, 38, 39, 40 Consequently, the differing clinical presentations, disease phenotype, and outcome are largely due to the stem cell stage at which these mutations occur, the type of driver mutations, the mutation load, the host genetic background, the complementing genetic events, and the order of acquisition of different mutations.41,42

Initial diagnostic evaluation depends on close correlation between laboratory data, clinical features, morphologic evaluation of PB and BM biopsy, and molecular genetic diagnostics. There is a wide spectrum in BM findings in PV, ET and PMF, with the most common morphologic features summarized in Table 3. As disease progresses, PV and ET show increasing fibrosis and osteosclerosis, largely as a result of abnormal megakaryocytic proliferation.43 It is critical to distinguish PV and ET from prefibrotic PMF, as patients with prefibrotic PMF have much higher risk of fibrotic and leukemic transformation, and shorter leukemia-free survival.44 Morphologic examination of a BM biopsy plays an important role in the differential diagnoses of pre-fibrotic PMF and PV/ET, detection of fibrotic progression, blast transformation, and secondary myelodysplastic syndrome (MDS)/AML. Use of CD34 immunostain is particularly useful for an accurate blast enumeration as the fibrosis in advanced stage disease precludes obtaining an adequate aspirate.45 European Myelofibrosis Network (EUMNET) has proposed a semi-quantitative grading system for serial monitoring of BM fibrosis.46 Recently, some researchers explored the role of quantitative polarization-sensitive mid-infrared spectroscopic imaging technology in the detection of disordered reticulin and collagen crosslinking, which may allow early recognition and more objective assessment of BM fibrosis and guide clinical decisions.47 For molecular analysis, sequential assessment of JAK2 V617F flowed by JAK2 exon 12 for suspected PV, or CALR and MPL for suspected ET or PMF, is a reasonable approach. However, an upfront NGS using a panel of genes including the three drivers and common co-operating mutations is highly recommended whenever possible, especially in patients who are negative for driver mutations (triple-negative) to detect non-canonical and other mutations to establish clonality. For follow-up monitoring, accurate quantification of driver mutations by highly sensitive RT-PCR or digital droplet PCR, with a sensitivity of 0.01%-0.001%, is recommended, especially in post-transplant setting.

Gene mutation profiling by NGS has identified co-operating mutations with prognostic and therapeutic value in MPN (Table 4). Unlike driver mutations, other co-operating mutations are not disease-specific and also occur in other hematopoietic and non-hematopoietic neoplasms, affecting genes involved in DNA methylation, histone modification, RNA splicing, transcription regulation, and signal transduction. These mutations play a role in modifying disease phenotype and contribute to disease progression. These additional mutations are more frequent in PMF and advanced disease compared to PV and ET. Some of these genes, such as ASXL1, EZH2, SRSF2, IDH1, IDH2, and U2AF1 Q157, have been referred as high molecular risk (HMR) mutations, and co-occurrence of two or more HMR mutations, along with absence of CALR type 1/type 1-like mutation, have been incorporated into prognostic scoring systems for PMF as adverse prognostic factors.48, 49, 50, 51, 52 The prognostic value of co-operating mutations has not been well-established in patients with post-PV or post-ET MF. However, it has been reported that approximately 50% of PV and ET patients also harbour non-driver mutations, the most frequent being ASXL1 and TET2.53 More importantly, the HMR mutations have been reported in 10% and 2% of ET and PV patients, respectively, and their incorporation into the genetic risk models (Mutation-enhanced International Prognostic Score System (MIPSS) ET and MIPSS PV) has improved risk stratification for these patients.54

More evidence that PV, ET and PMF belong to the same disease spectrum come from the common cytogenetic changes. Cytogenetic aberrations are found at diagnosis in 15-20% of patients with PV and 5-10% of patients with ET, with del(20q), +8, and +9 being the most common.55,56 Cytogenetic changes are much more common in PMF, seen in 15-20% of prefibrotic PMF and 30-40% of overt PMF cases, and include the same changes with the addition of del(13q), der(6)t(1;6)(q21-23;p21.3), and 1q21 rearrangements.57,58 Some of these changes, including complex karyotype or ≥1 abnormality in +8, 7/7q-, i (17q), 5/5q-, 12p-, inv(3), or 11q23 rearrangement, have been associated with an increased risk of leukemic transformation and regarded as unfavorable factors in the Dynamic International Prognostic Scoring System (DPISS).59

Patients with MPN have an inherit predisposition to transform to MPN-BP. The incidence of leukemia at 10 years is 2-15% for PV, 1-3% for ET, and 10-30% for PMF.60,61 Other risk factors for leukemic transformation include advanced age, severe anemia, thrombocytopenia, leukocytosis, blast count >5%, increasing BM fibrosis, lack of driver mutations, absent type 1/type 1-like CALR mutation, ≥2 HMR mutations, and adverse cytogenetics.62 In addition, it has been reported that EZH2 inactivation and NRAS mutation cooperate to induce leukemic transformation of MPN by enhancing mTOR signalling.63 AML arising from MPN has different biology and genomic profile compared with de novo AML, and is characterized by a distinct mutation profile (higher frequency of IDH1, IDH2, JAK2, SRSF2, TET2 and TP53 mutations, rarity of DNMT3A, FLT3 and NPM1 mutations)64, 65, 66 and dismal prognosis, with a median overall survival of less than 6 months without treatment.65, 66, 67 Studies on paired BM samples from CP and BP have shown both acquisition and loss of mutations during clonal evolution, suggesting that the dominant clones in BP may arise from the same founder clone or unrelated clones. The fact that AML is increased in those treated with cytotoxic agents and lack of JAK2 mutation in many of these therapy-related AML support secondary leukemia rather than MPN progression in some cases.68,69 The effect of increasing BM fibrosis and the resulting ineffective BM microenvironment on driving genetic instability is suggested.

Chronic neutrophilic leukemia (CNL) is a rare subtype of MPN with well-established molecular abnormality and is characterized by persistent absolute neutrophilia (white blood cell count ≥25×109/L, with ≥80% segmented neutrophils and bands, and <10% neutrophil precursors), hypercellular BM with mature granulocyte proliferation, and hepatosplenomegaly. It has been shown that 80-90% of CNL patients harbour mutations in CSF3R.70, 71, 72 The majority of patients carry a point mutation in the membrane proximal region (with T618 in exon 14 being the most common) that leads to JAK-STAT activation and good response to ruxolitinib, a JAK inhibitor, whereas a smaller subset of patients have nonsense or frameshift mutations in exon 17 resulting in truncation of the cytoplasmic tail and SRC-TNK2 kinase dysregulation and sensitivity to dasatinib, a SRC kinase inhibitor.70,73, 74, 75 Secondary gene mutations have been detected in some CNL patients, such as ASXL1, TET2, SETBP1, SRSF2, and U2AF1, but without well-established prognostic significance.76, 77, 78 Recently, it is suggested by the ICC of Myeloid Neoplasms that the key diagnostic threshold for leucocytosis should be lowered to ≥13×109/L in cases with activating CSF3R mutations.3 The prognosis of CNL remains relatively poor, with most patients succumb to complications or undergo leukemic transformation.79

Eosinophilia has the most complex workup among all MPN and may arise as a result of reactive, neoplastic, or idiopathic conditions. Reactive causes, such as drug reaction, allergic state, parasite infection, and T-cell lymphoma or Hodgkin lymphoma, account for most cases. Common hematopoietic neoplasms associated with eosinophilia include myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (M/LN-eo-TK, such as rearrangements of PDGFRA, PDGFRB, FGFR1, JAK2, or FLT3, or ETV6::ABL1 fusion), eosinophilia associated with myeloid neoplasms, somatic mastocytosis (SM), and chronic eosinophilic leukemia (CEL). In eosinophilia associated with hematopoietic neoplasms, eosinophils often have the same molecular genetic abnormalities as the neoplastic hematopoietic components. Idiopathic hypereosinophilia syndrome (iHES) is defined as persistent PB eosinophilia (≥1.5×109/L, ≥6 months) with associated tissue/organ damage due to cytokines/enzymes released from eosinophils, without definite reactive causes or evidence of clonality, Cases with persistent hypereosinophilia of unknown etiology without tissue/organ damage is referred to as idiopathic hypereosinophilia (iHE) or hypereosinophilia of unknown significance (HEus).80,81 (Figure 1)

Chronic eosinophilic leukemia is characterized by clonal proliferation of morphologically abnormal eosinophils resulting in persistent eosinophilia (≥1.5×109/L) in PB, BM and peripheral tissues, with ≥10% eosinophils, and tissue/organ damage. The diagnosis requires exclusion of other genetically defined entities, such as AML with CBFB::MYH11, AML with RUNX1:RUNX1T1, CML, chronic myelomonocytic leukemia (CMML), M/LN-eo-TK, and SM.80 Recently, both the 5th edition of WHO and ICC have included morphologic dysplasia, especially in megakaryocytes, in the definition of CEL.1,3 The advent of NGS has led to the detection of mutations in a significant subset of patients with eosinophilic disorders who would previously be classified as iHES.82, 83, 84 The most commonly mutated genes included ASXL1, TET2, EZH2, SETBP1, CBL, NOTCH1, DNMT3A, SRSF2 and TP53.82,83 More recently, activating STAT5B N642H mutation and JAK1 mutation have been identified in a small subset of patients with eosinophilia.85,86 The detection of these recurrent mutations or karyotypic abnormalities (such as trisomy 8, deletion 7, iso17q) provides evidence of clonality and supports a diagnosis of CEL. As a result, the 5th edition of WHO has dropped the criteria of increased blasts (≥2% in PB or 5-19% in BM) as a surrogate of clonality.80 Moreover, identification of genetic aberrations has therapeutic implications.87 However, it is of note that some mutations have been reported in aging individuals as “CHIP” mutations (such as DNMT3A, TET2, and ASXL1), in particularly when they are present as a single mutation and/or at a low VAF,88,89 thus should be interpreted with caution.

Juvenile myelomonocytic leukemia (JMML) is a MPN of early childhood that is characterized by leukocytosis with absolute monocytosis (≥1×109/L), splenomegaly, and constitutive activation of RAS signaling pathway. Approximately 90% of cases involve driver mutations in one of five genes in the RAS/RAF/MEK/ERK axis, PTPN11 (35%), KRAS/NRAS (25%), NF1 (15%), and CBL (15%).90, 91, 92, 93 The 5th edition of WHO classification has listed JMML under MPN in order to emphasize its molecular pathogenesis and recognize the absence of significant morphologic dysplasia.1 It is of note that ICC has listed JMML under the category “pediatric and/or germline mutation-associated disorders”.3

Mutations in JMML can occur either as somatic or as germline, and different mutations are associated with different disease phenotypes, ranging from cases with aggressive clinical course and dismal prognosis (such as cases initiated by somatic PTPN11 mutations or germline NF1 mutations)90,92,94 to cases with indolent clinical course that may spontaneously revolve (such as cases associated with germline CBL mutations).95,96 Somatic mutations in PTPN11, NRAS and KRAS are characterized by heterozygous gain-of-function mutations in children without well-defined genetic syndromes, whereas germline mutations in NF1 and CBL are often seen in children with genetic syndromes (neurofibromatosis type 1 and CBL syndrome, respectively), with progression to JMML upon loss of the normal allele, as the result of uniparental disomy or somatic mutations, insertions, or deletions.97, 98, 99 Around 10% of JMML patients do not carry any canonical mutations in RAS pathway. Among these cases, gain-of-function somatic mutations in the RRAS or RRAS2 gene,100 or ALK and ROS1 tyrosine kinase fusions,101 have been identified as initiating events in a small subset of patients.

Secondary mutations are seen in approximately 50% of JMML patients, either affecting another gene in RAS pathway, such as double KRAS/NRAS mutations, or involving genes outside RAS pathways, with SETBP1 being the most common.102 Other recurrent secondary mutations include ASXL1, JAK3, SH2B3, EZH2, DNMT3A, RUNX1, GATA2, and ZRSR2.100,102,103 These cooperating mutations are usually subclonal in nature, and have been associated with disease progression and an aggressive clinical course.100,101,104 Moreover, genome-wide DNA methylation profiling has identified DNA methylation signatures to be correlated with genetic aberrations and clinical outcome, with the high methylation group more enriched in cases with PTPN11 mutations and having a high risk of relapse after hematopoietic stem cell transplantation, the low methylation group more enriched in cases with NRAS or CBL mutations and having a favorable prognosis, and the intermediate methylation group more enriched in KRAS mutations and monosomy 7.105, 106, 107

It is recommended that the molecular work-up for patients with suspected JMML should include NGS testing using a panel of genes encompassing the 5 canonical mutations and other secondary mutations, on both tumor and normal germline samples. Routine karyotypic analysis and FISH for monosomy 7 should also be performed. Approximately 30-40% of JMML patients have cytogenetic aberrations, most commonly monosomy 7, seen in 25% of patients.94

Myeloproliferative neoplasm, unclassifiable (MPN-U) is a heterogeneous group of MPN that should be restricted to cases with clinical, laboratory, morphologic, cytogenetic and molecular features of MPN, but cannot be classified into any of the above specific MPN subtypes due to not meeting sufficient diagnostic criteria or displaying overlap features. Patients may present in an early-phase when the diagnostic features are not fully developed or at a late-stage with significant myelofibrosis, osteosclerosis, dysplastic changes, and increased blasts that mask the underlying disease. Patients with MPN and co-existing inflammatory or neoplastic disorders that obscure some of the diagnostic features are also included in this category. There are no specific molecular genetic aberrations. It is recommended that the clinical work-up, especially for patients presenting with early-stage disease, requires a dynamic and integrated approach and close monitoring to identify features that may allow re-classification into other specific MPN type.108

留言 (0)

沒有登入
gif