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Atypical CML- the role of morphology and precision genomics
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Atypical CML- the role of morphology and precision genomics Terra Lasho Division of Hematology, Mayo Clinic Rochester, USA
A R T IC LE I N F O
ABS TRA CT
Keywords: Atypical chronic myeloid leukemia Clonal expansion Precision genomics Dysplastic granulopoiesis Clonal evolution
Atypical chronic myeloid leukemia is an esoteric myeloid malignancy with features of both myeloproliferative and myelodysplastic syndromes. This disease is characterized primarily by morphologic-based criteria, and has clinical and molecular features overlapping with other myeloid malignancies. No one molecular abnormality is specific, and multiple mutations are often present in various combinations, due to the malignant multi-step clonal evolution of myeloid malignancies. In this review, we will address what we know about atypical chronic myeloid leukemia; evaluate how the molecular landscape in myeloid malignancies overlaps, and discuss what we can learn by incorporating individualized precision genomic strategies.
Introduction Atypical chronic myeloid leukemia (aCML) is a rare clonal hematopoietic stem cell disorder with absence of a detectable BCRABL1 fusion or Philadelphia (Ph) chromosome. It is currently recognized by the WHO 2016 revised classification as a sub-type of a myelodysplastic/myeloproliferative (MDS/MPN) overlap syndrome, as it displays both proliferative (neutrophil leukocytosis) and dysplastic (prominent granulocyte dysplasia) features [1–3]. The estimated incidence of aCML is 1% (or 1 out of every 100) of Phpositive CML cases with a median overall survival of ~25 months (range, 14–30 months) [4–7]. Clinical presentation is similar to other known myeloid malignancies and includes anemia, splenomegaly, and thrombocytopenia. Patients typically present in the sixth and seventh decade of life and there is a slight male predominance. The normal disease course typically ends in complications from cytopenias, or transformation to acute myeloid leukemia (AML) in 30–40% of cases [4,6,8,9]. The etiology of aCML is not known and the current diagnostic criteria for aCML are a result of a stratification of an accumulation of morphological similarities, which are the most important distinguishing features for diagnosing atypical CML. From what we can tell, the molecular landscape of aCML is heterogeneous. Molecular features include an increased frequency of gene fusions or aneuploid karyotype and/or recurrent somatic mutations affecting genes involved in growth factor signaling, transcriptional regulation, RNA splicing, and epigenetic regulation of DNA methylation/histone modification [6,10,11]. Methods for detecting genetic alterations in aCML have varied as sequencing techniques have rapidly evolved in scope and sensitivity. This along with the paucity of this disease has made molecular profiling difficult. There is no one genetic alteration that is specific to the disease, and in fact, all described genes with pathogenic mutations in aCML have been recognized in related myeloid malignancies. This is conceivable, because along with other myeloid malignancies, aCML contains various combinations of genetic abnormalities due to the malignant multi-step clonal evolution of the disease. The influence of a gene mutation on clinical presentation may not be straightforward and can be due to mutation-specific functional effects, and/or be co-influenced by other mutations, copy number or cellular prevalence of mutations, and the order of mutant acquisition. Ultimately, this challenges the management of, and underscores the marked clinical heterogeneity in these disorders. In this review we will evaluate the clinical parameters in aCML, management, and treatment. We will discuss why there is such
E-mail address: [email protected]. https://doi.org/10.1016/j.beha.2019.101133 Received 2 December 2019; Accepted 3 December 2019 1521-6926/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Terra Lasho, Best Practice & Research Clinical Haematology, https://doi.org/10.1016/j.beha.2019.101133
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clinical variability in myeloid malignancies, and why there is a critical need for a precision genomics-based approach in clinical practice, especially in an esoteric disease like aCML. Precision genomics refers to an individualized approach to understanding molecular pathogenic contributions in disease by taking into account differential influence of varying aspects of genetic alterations. Instead of a “one size fits all” approach, this method provides an individualized mutant genetic/functional summary which has the ability to be translated into a tailored targeted therapy. Background Historically, aCML was considered to be a myeloproliferative subtype of chronic myeloid leukemia (CML) and was first given the name “CML-like syndrome”, because both diseases can present with splenomegaly and display myeloid hyperplasia with left-shifted peripheral granulopoiesis. However, aCML displays some distinct features (primarily no BCR/ABL, poorer survival, and granulocytic dysplasia), and so was subsequently considered an intermediate syndrome somewhere between CML and chronic myelomonocytic leukemia (CMML) on the spectrum of disorders [12]. In 1994, the French-American-British (FAB) Cooperative Leukemia Group studied fifty-one cases of BCR-positive/negative cases and recommended the implementation of measures to distinguish aCML from CML and CMML [13]. These criteria included five quantification parameters (complete white blood cell count [WBC], percentage immature granulocytes, percentage monocytes, percentage basophils, percentage erythroid precursors in bone marrow) and one qualitative parameter (granulocytic dysplasia). During the next couple of years, there were reports supporting the FAB criteria [14], some soliciting the need for better criteria definitions and some claiming that cases satisfied multiple criteria. This raised the question of whether to consider aCML as its own entity, part of a spectrum of CMML, or an unusual evolution of MDS [15–17]. However, in 2001, the WHO created a new disease classification, myelodysplastic (MDS)/myeloproliferative neoplasm (MPN) overlap syndrome. This categorization helped to further differentiate aCML from CML. In 2008, the WHO further distinguished aCML by including a numeric threshold of leukocytosis (≥13 × 109/L) and monocytes (< 10% of WBC), absence of PDGFRA, PDGFRB, and FGFR1 rearrangements, and presence of dysplasia in the granulocytic lineage. In the most recent revision to myeloid malignancy diagnostic criteria, the 2016 WHO revised the criteria for aCML (See Table 1) which included an emphasis on molecular alterations. This includes stratification by presence of SETBP1 and ETNK1 mutations, and if CSF3R mutations are present the case should be rigorously reviewed to exclude alternate diagnosis of chronic neutrophilic leukemia (CNL). (See Fig. 1). Laboratory findings Peripheral blood (PB) WBC counts in aCML are higher than 13 × 109/L (typical range: ~35–96 × 109/L) [4,14]. Immature neutrophils (including promyelocytes, myelocytes, and metamyelocytes) account for ≥10–20% of WBC, but blast percentage is usually less than 5% and always less than 20%. The morphology of neutrophils may be abnormal and predominantly include one of more: hypogranular cytoplasm, exaggerated clumping of nuclear chromatin, and abnormal lobated nuclei (pseudo-pelger huet). Basophilia is usually minimal (≤2%) or absent [4,12,13]. Evidence of dyserythropoiesis is also commonly observed (example is macro-ovalocytosis). However, the most important feature when examining the peripheral blood are the levels of monocytes. This important criterion distinguishes aCML from CMML. In aCML, monocytosis may be present, but is always less than 10% of the WBCs. The bone marrow (BM) in aCML is hypercellular and includes a predominantly granulocytic proliferation. Granulocytic dysplasia is often noticeable with similar features to the PB. Other lineages may be dysplastic as well, with dyserythropoiesis present in up to 50% of cases [4,14]. Staining for CD34 + helps to reveal blasts which may be present, but should not be quantitatively over 20% of all nucleated cells. Although bone marrow histopathology reveals overlap between aCML and CMML which can sometimes be hard to differentiate, dual staining of esterase can distinguish monocytes in the BM biopsy using cytochemical staining and can be helpful for ruling out CMML (See Fig. 2). Overview of clonal evolution of myeloid malignancies Myeloid malignancies arise from an altered hematopoietic stem cell with an acquired influence over the growth and differentiation capacity of one or more cell lineages in the bone marrow and blood. This evolution progresses as a stepwise accumulation of Table 1 WHO Diagnostic Criteria for aCML WHO Diagnostic Criteria (2016) Atypical Chronic Myeloid Leukemia due to increased numbers of neutrophils and their precursors (promyelocytes, myelocytes, metamyelocytes) comprising ≥10% of leukocytes • Leukocytosis which may include abnormal chromatin clumping • Dysgranulopoiesis, bone marrow with granulocytic proliferation and dysplasia, with or without dysplasia in erythroid and megakaryocytic lineages • Hypercellular and bone marrow blast count of less than 20% • Blood of BCR/ABL fusion gene, or rearrangement of PDGFRA and B, FGFR1 or PCM – JAK2 • Absence or minimal absolute monocytosis (usually < 10% of leukocytes) • No • No or minimal basophils (usually < 2%)
2
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Fig. 1. Morphological stratification for MDS/MPN overlap syndromes. After ruling out a secondary cause, AML, and other fusions, identification for CMML, RARS-T, aCML, and MDS/MPN-U is provided by the corresponding flow chart path. Highlighted in green are several genes which, when observed as mutated are additional indicators of disease type.
Fig. 2. Blood and bone marrow of aCML, BCR-ABL1 negative. A). Wright-giemsa stain (100x) of peripheral blood showing elevated white blood cell count, immature myeloid cells (red arrows) and dysplastic granulocytes (black arrows). B). Wright-giemsa stain (400x) of peripheral showing pseudo-Pelger-Huet cell (black arrow). C). Wright-giemsa stain (200x) of bone marrow aspirate demonstrating granulocytic dysplasia (black arrows). D). Hematoxylin eosin stain (100x) of bone marrow biopsy indicating hypercellularity (involving granulocytic proliferation and dysplasia).
gene mutations which promotes a multi-lineage selective advantage of mutant clonal expansion over normal hematopoiesis. The result is clonal expansion which encourages evolution of disease and consequential complex effects on the genetic, epi-genetic, and microenvironment (See Fig. 3). Appreciating this clonal architecture is essential to understanding how a relatively small group of known gene mutations can orchestrate the extensive clinical and molecular overlap reflected in the proliferative and dysplastic features of myeloid malignancies at diagnosis and throughout the course of disease. A functionally well-characterized gene mutation may influence the transcriptome or epigenome differently in the presence of additional mutations and the type of association as well as the order of mutations have an impact on the kinetics of disease development. We can determine the order of mutant occurrence by assessing mutant variant allele frequencies (VAF) –which is a measure of the ratio of mutant to wild-type allele frequency in a sample, or performing clonogenic assays using BM or PB to expand individual hematopoietic colonies (sub-clones) representing the spectrum of clonal architecture. A 3
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Fig. 3. Clonal evolution influence on disease. aCML is the result of an aberrant clonal expansion. As seen in the top panel, acquisition and propagation of mutations in a cell population promotes expansion and additional mutations which outcompetes normal hematopoiesis. Below is a list of how a disease phenotype can be affected or influenced through clonal evolution.
critical observation in myeloid malignancies is that most of the key genes that are mutated are not specific to a single disease subtype, and in addition, most of the recognized mutated genes can be attributed to ~4 gene function groups: pre-mRNA splicing (SRSF2, U2AF1, ZRSR2 and SF3B1), growth factor signaling transduction (RAS-pathway, CSF3R, JAK2, CALR, MPL, FLT3, KIT, ETNK1), transcription factor (TF)/or TF regulation (RUNX1, CEBPA, GATA2, NPM1), and chromatin and epigenetic regulation of DNA methylation and histone modification (ASXL1, EZH2, BCOR, SETBP1) [10,18]. As the genetic hierarchy of disease is defined, associations can be easier to predict regarding clinical and genetic classifications. We have learned a lot about this from several large scale single-cell based analyses and clonal reconstruction datasets, involving acute myeloid leukemia (AML) [19–22]. One important observation is that mutations involving the genes DNMT3A, ASXL1, and TET2, are not only important prognostic indicators of disease, they have been shown to appear years before any sign of disease, and are considered CHIP genes “clonal hematopoiesis of indeterminate potential” because of their prevalence in healthy people > 50 years of age. The incidence of acquiring mutations in these genes rises with age, and they are associated with risk of development of a blood disorder [23]. Mutations in CHIP genes persist in cases of relapse and remissions, and their presence have been shown to provide a selective multi-lineage advantage to mutant over wild-type hematopoietic progenitor cells [24–26]. Because of this, they are considered “pre-leukemic” and important for disease initiation. CHIP mutations are almost always associated with other recurrent mutations, suggesting functional cooperation in acquisition/progression of leukemia as observed in AML cohorts as well as in other myeloid malignancies. However, their existence is not critical for disease [27,28]. CHIP genes may or not present as CHIP and are gene members of one of most recognized mutated gene functional categories: chromatin and epigenetic regulation. Almost 90% of de novo AMLs with a normal karyotype follow a classical hierarchy of acquisition of mutations, which involves a successive occurrence of a mutation in epigenetic regulation including DNA methylation, followed by a mutation in a TF, then finally, a mutation involving signaling/tyrosine kinase pathway [19,20,22,29]. In a study of one thousand five hundred and forty AML cases, the order of mutations (identified by using variant allele frequencies) revealed CHIP mutations first (TET2 [16%] and DNMT3A [54%]), followed by TF mutations (NPM1), and finally signaling mutations (FLT3 [39%], NRAS/PTPN11 [34%]). A second clonal hierarchy has been described in de novo AML which excludes CHIP mutations, but keeps the TF (RUNX1, GATA2, CEBPA) and then signaling gene acquisition order [21]. This is important because it provides a kind of blueprint toward the evolution of a myeloid malignancy, that is, epigenetic changes which promote or at least provide a background for transcriptional/signaling dysregulation, affecting differentiation, proliferation, and survival of cell types. Interestingly, in secondary AML, which frequently arises from MDS (in more than 20% of cases), the acquisition of mutations differs, and instead involves splicing genes (SRSF2, SF3B1, U2AF1, and ZRSR2) and chromatin modifiers (BCOR, ASXL1, EZH2). In a study of one hundred ninety-four cases of either secondary or therapy-related AMLs, the two most frequent mutated genes involved ASXL1 (32%) and SRSF2 (20%) [20]. Sixty-three percent of SRSF2-mutated s-AML are also mutated with ASXL1. This is significant because ASXL1 mutations are CHIP-related, and patients with MDS are of advanced age, so it is logical that they are present. Splicing mutations appear to be associated with the pathogenesis of myelodysplasia as in a study of nine hundred and forty-four cases of MDS they represent the most mutated functional gene group (mutated in 64%) [30]. The six genes mutated in more than 10% 4
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of MDS cases included: TET2, ASXL1, SRSF2, SF3B1, DNMT3A, and RUNX1. In several other smaller studies, the most recurrent mutant gene associations in MDS were between IDH2, STAG2, ASXL1, RUNX1, and BCOR, with ASXL1 being the most frequent [31–33]. TET2 mutations were associated with SRSF2 mutations, and this is very significant for CMML also, where the thought of early clonal dominance of TET2 mutated cells have the ability to skew myeloid differentiation toward the granulomonocytic lineages (possibly elucidating the CMML/monocytosis relationship) [34]. Overall, VAF analysis in MDS demonstrated that mutations in DNA methylation and splicing appear before all others and mutations in signaling pathways occurred last, and this has been verified in multiple studies [31,32,35,36]. Finally, in BCR-ABL-negative MPNs which have evolved to blast phase, (1% in essential thrombocythemia [ET], 7% for polycythemia vera [PV], and 20% in primary myelofibrosis [PMF]), mutations up-regulating the JAK/STAT-signaling pathway (CALR, and MPL) appear first followed by mutations in chromatin modifiers (TET2, ASXL1 or DNMT3A). In PMF, JAK2 mutations appear first followed by a mutation in chromatin modifiers (ASXL1 or EZH2). However, in ET, mutations in JAK2 occur as a sub-clonal event to TET2, DNMT3A, or SF3B1 [37,38]. This demonstrates that the order of mutations is important to identify as this can influence the differential diagnostic criteria for the development of disease (in this case, PMF vs. ET, which includes the ratio of granulocyte and erythroid expansion and atypical features of megakaryocytes). Finally, the allele frequency of a JAK2V617F mutation can change (increase in VAF), and this can influence the disease enough to change the diagnosis from ET to PV [39]. Whether this is due to the increase in clonal burden of JAK2V617F due to VAF or gene dosage, or a result of pre-existing or additional somatic mutations, reinforces the need to completely characterize the mutational environment in order to have an effective perspective of molecular pathogenesis. Overall, it is critical to understand that not only the function of a mutation, but the order of acquisition, VAF, cellular presence (gene dosage), or co-expression with other mutations can influence the pathophysiology and disease clinical presentation in myeloid malignancies. Unlike CML, which is clearly defined by the consistent presence of BCR-ABL1 fusion, we have a limited understanding of aCML pathobiology because of the lack of specific molecular markers and overlapping morphologic features with other MDS/MPN syndromes, which also include: MDS/MPN-U and CMML. According to the International MDS/MPN Working Group, although “there have been a number of contributions to literature, the MDS/MPN overlap syndromes have considerable molecular heterogeneity, and lack of specific markers” [40]. Just recently, the largest study based on genetic-assessment of MDS/MPN overlap syndromes was published. In this study, whole exome sequencing was performed on one hundred and fifty-eight patients which included MDS/MPN overlap syndromes (including n = 27 aCML) [41]. Unlike previous studies where disease sub-types were initially stratified by WHO criteria and then individually screened and compared for molecular alterations, this group performed an unsupervised mutational screening to then stratify disease types by mutation profile. The overall findings revealed a complex overlap among genetic mutations and disease. No single mutation or combination of mutations defined a singular disease sub-type. In essence, the findings highlight the idea that dose and sequential effect of clonal evolution, and the order of occurrence contribute to the heterogeneity of the clinical features within this group of diseases. This helps to explain why there is such extreme difficulty in clarifying diagnostic classifications based solely on histology for some cases. What do we do with this information? Well, (in addition to understanding the mutational co-influence perspective from the AML studies above) this report did reveal some interesting mutational relationships which correlated to clinical features. They reported that more than half of the patients demonstrated co-occurring mutations in at least three different gene functional pathways. And in the majority of cases, by examining the VAF structure, they provide evidence that patients had at least two mutations involving epigenetic regulation and splicing factors and these were in the dominant clone, with acquisition of additional mutations. We know that splicing mutations are frequent in MDS. They also observed that mutations in ASXL1, TET2, and EZH2 correlated with older age (consistent with what we discussed above as ASXL1 and TET2 are CHIP-related genes). Mutations in CSF3R, which is a cytokine that controls the regulation of granulocytes, were (of course) associated with high neutrophil count, but also presence of splenomegaly and low monocyte count (CSF3RT618I mutations have been reported as highly associated to CNL [42]). The presence of signaling mutations, NRAS in particular, were associated with low hemoglobin, platelet count, and neutrophil percentage, and high monocyte percentage. ASXL1 mutations were associated with high WBC and need for platelet transfusion. TET2 mutations were associated with low neutrophil percentage, high monocyte percentage, BM dysplasia, low platelet count, and consistently high platelet transfusion requirement. And finally, SETBP1 mutations were associated with high hemoglobin level and high platelet count. In addition to singular mutations, aberrant karyotypes and copy number variations were also detected in these cases. Molecular landscape of aCML For aCML, the most frequent mutations described (to varying degrees) have been found in SETBP1, ASXL1, (N/K)RAS, SRSF2, TET2, CBL, CSF3R, and ETNK1 [6]. [10,11,18,40,43–47] However, the comparison of longitudinal studies is limited to the range and depth of the technique applied (See Table 2). Of these, mutations in SETBP1 and ETNK1 appear to be the most recurrent genes mutated in aCML and are seen in up to a third of patients [45]. [48,49] Mutations in SETBP1 in fact have been reported to have a seven-fold lower incidence in CMML and uncommon occurrence in MPN, suggestive of this as a potential molecular marker a useful diagnostic tool to differentiate aCML between these clinically similar entities (and represented as a mutation of diagnostic emphasis for aCML by WHO-2016 revised criteria) [45,49,50]. Multiple studies have also shown that mutated SETBP1 is associated with a more adverse clinical presentation (higher leukocyte count, lower hemoglobin level, thrombocytopenia), suggesting this mutation is not only relevant to leukemic oncogenesis, but also provides important prognostic value [45,49,51]. There is also a strong correlation of mutated SETBP1 with mutations involving the chromatin remodeling gene, ASXL1, a 5
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Table 2 Mutational Gene Spectrum in aCML Type
Gene
Name
Frequency (%)
Reference
Epigenetic regulation of DNA methylation/ histone modification
IDH1/IDH2
Isocitrate Dehydrogenase 1 (cytosolic), Isocitrate Dehydrogenase 2 (Mitochondrial) Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit Additional Sex Comb Like 1 SET Binding Protein 1 Tet Methylcytosine Dioxygenase 2 Serine And Arginine Rich Splicing Factor 2 U2 Small Nuclear RNA Auxillary Factor 1 Splicing Factor 3b Subunit 1 Zinc Finger CCCH-Type, RNA Binding Motif And Serine/ Arginine Rich 2 N/K Proto-Oncogene Receptor, GTPase Janus Kinase 2 Colony Stimulating Factor 3 Receptor (Granulocyte) Calreticulin MPL Proto-Oncogene, Thrombopoietin Receptor KIT Proto-Oncogene Receptor Tyrosine Kinase Fms Related Tyrosine Kinase 3 Fms Related Tyrosine Kinase 3 - internal tandem duplication Cbl Proto-Oncogene Ethanolamine Kinase 1 Protein Tyrosine Phosphatase, Non-Receptor Type 11 Runt Related Transcription Factor 1 CCAAT Enhancer Binding Protein α Nucleophosmin 1 IKAROS Family Zinc Finger 1
0 to 4
[6,52]
8 to 20
[48,52]
20 to 66 12 to 33 16 to 41 12 to 40 0 to 20 8 4
[6,49,52] [6,11,45,48,52] [11,52] [11,52] [48,52] [52] [52]
8 to 3 to 0 to 0 to 0 to 0 7.1 4
[6,9,48,52] [6,11,52] [6,10,11,52] [6,11,47,52] [6,11,52] [6] [6] [52]
EZH2 ASXL1 SETBP1 TET2 SRSF2 U2AF1 SF3B1 ZRSR2
RNA splicing
Signal Transduction
(N/K) RAS JAK2 CSF3R CALR MPL KIT FLT3 FLT3-ITD CBL ETNK1 PTPN11 RUNX1 CEBPA NPM1 IKZF
Transcription Factor
35 8 40 4 2
0 to 10 8 to 8.8 4 12 11.8 0 0
[11,48,52] [46,52] [52] [52] [6,52] [6,52] [52]
commonly mutated oncogene with detrimental prognostic impact, suggesting their potential cooperative function in leukemic progression of aCML. In a cohort of 39 cases, co-mutated SETBP1 and ASXL1 occurred in 48% of aCML patients. ASXL1 has already been shown to be a high risk prognostic marker in AML, MDS, and PMF, and may also provide further prognostic value in aCML [11,49,53]. However, mutations in ASXL1 were not shown to affect overall survival in a study by Patnaik et al. [52]. In 2015, recurrent mutations in ETNK1 were found at a prevalence of 8.8% in sixty-eight cases of aCML. All mutations were heterozygous, and in two cases were present concurrent with SETBP1 mutations [48]. The ETNK1 gene protein functions as a kinase in phospholipid biogenesis. The exact functional role of mutations in this gene is not yet clear, however, mutations appear to be relatively specific to aCML, CMML, and systemic mastocytosis with associated eosinophilia [48,54]. Although cases of aCML have myeloproliferative features, including splenomegaly and leukocytosis, mutations in the most wellknown driver genes found in myeloproliferative disorders (JAK2, MPL, and CALR) are typically not found in aCML. However, JAK2V617F has been cited with low frequency (~4–8%) [6,18,55]. This is an important observation suggesting that this type of mutation is unlikely to be a driver mutation in aCML, but is still valuable in differentiating the MDS/MPN overlap syndromes from MPN [56,57].
Cytogenetic Karyotypic abnormalities in aCML range widely and have been reported in 20–80% of patients. The most common abnormalities are +8 and del (20q), but abnormalities of chromosomes 13, 14, 17, 19 and 12 are also commonly reported [9]. There was a report of Trisomy 14 in a case of aCML in 1990 [58]. Several case studies resulting in fusions have also been reported [59–62]. Sequencing detection methods are now becoming available, including RNAseq, which can interrogate the transcriptome and detect the presence Table 3 Other Genetic Alterations in aCML Type
Chromosomal abnormality
Most common
+8 and del (20q) abnormalities of chromosomes 13, 14, 17, 19 and 12 Trisomy 14 46XX,t(2; 13; 2;21)(p13; q12; q33; q11.2) t(3; 21)(q22; q22) t(1; 9)(p34; q34) t(7; 11) (p15; p15)
Case Case Case Case Case
Report Report Report Report Report
Fusion Gene
6
Reference [9]
SPTBN1-FLT3 RYK/ATP5O CSF3R/SPTAN1 NUP98-HOXA9
[58] [59] [60] [61] [62]
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of aberrant fusion genes. Therefore, the presence/identification of fusion genes may become more important and specific as more cases are probed (See Table 3). Prognostic factors and treatment Atypical CML has an aggressive disease course, with a poor prognosis and a median survival of ~25 months (range, 14–30 months) [4–6]. Clinical prognostic factors included age > 65 years, female, hemoglobin < 10 g/dL, WBC count > 50 × 109/L, and immature circulating precursors. In addition, mutations in ASXL1 and SETBP1 have been associated with high risk [63]. No current standard of care exists for aCML. Because of the short predicted overall survival of the disease, there is no prognostic algorithm advising aggressiveness of treatment. Constitutive symptoms as well as anemia, thrombocytopenia, progressive leukocytosis, and symptomatic splenomegaly should be managed if present with standard supportive care measures used currently for MDS and MPN. Allogeneic stem cell transplant appears to be the only long-term treatment option; however, most patients are not eligible due to advanced age at diagnosis and comorbidities. Additional current treatment options include hypomethylating agents, which have demonstrated transient improvement in clinical symptoms [7,64]. Rarely, complete and partial responses have been reported with hydroxyurea, although short lived [8,65,66]. Hydroxyurea can also be used for supportive care in the presence of another treatment to control progressive leukocytosis or splenomegaly. Standard IFN-α therapy has been described with varying durability of response [8,65,66]. In a phase 2 study of PEFIFN-a-2b, two aCML patients out of five achieved a complete remission after only 3 months of therapy. Both patients were discontinued due to toxicity; however, the median duration of therapy was 36 and 38 months. This type of therapy will need to be further assessed [67]. However, the most interesting and promising therapeutic options include targeted therapies currently available for specific mutational profiles. This approach will be based on the ability to utilize an individualized precision-genomic-based approach to assess genetic/functional profiles in aCML (and related myeloid malignancies). Therefore, for practicing clinicians now, it is essential to molecularly screen incoming patients using available myeloid mutation genotyping panels not only to better understand the contribution of these mutations to the pathobiology, but to help to identify targets for individualized gene-targeted therapy [64,68–74]. Summary and future directions Atypical CML is heterogeneous, both clinically and molecularly, to other myeloid malignancies, especially MDS/MPN overlap syndromes. This is partly due to the difficulty in comparing reports from a longitudinal characterization over several decades of redefined diagnostic criteria/emerging sequencing techniques. However, the mutated gene profile in aCML is similar to other MDS/ MPN overlap syndromes and additional myeloid malignancies. Large scale clonality studies have demonstrated that the function of a specific mutation may be less important than its order of acquisition, allele frequency or copy number, and/or co-influence with other mutations, in determining the pathobiology of a disease. This is well described in large AML single-cell and clonal studies, where the order of acquisition of types of mutation relates to disease clinical presentation and progression. These studies have also verified that splicing mutations are associated to myelodysplasia, and early acquisition of TET2/SRSF2 mutations influences the granulomonocytic predisposition in CMML and may drive the characteristic feature of monocytosis that distinguishes CMML from other MDS/MPNs. Precision genomics is a paradigm changing method to individually approach disease on a case by case basis, by taking into account differential influence of varying aspects of genetic alterations. Instead of a “one size fits all” approach, this method provides an individualized mutant genetic/functional summary which has the ability to detect early and distinct phases of disease, and be incorporated into a tailored targeted therapy. This ability has already demonstrated promising strategies in aCML, including the recent proposal of considering the reactivation of PP2A as a therapeutic strategy in SETBP1-mutated cells [75]. Practice points
• There is a current lack of understanding of the molecular landscape in aCML. • Practicing clinicians must utilize available myeloid screening panels to interrogate cases of aCML –This will help identify targets for individualized therapy • Push to incorporate Precision Genomics into practice: o o o o
Paradigm change from “one-size-fits-all” to individualized fingerprint as a diagnostic strategy Individualized case assessment by merging deep screening techniques Ability to detect early and distinct phases of disease Pharmacogenomics -provide individualized targeted therapy “personalized medical cocktail”
Research agenda
• Etiology of aCML is not well understood • Diagnostic criteria for aCML are a result of a stratification of an accumulation of morphological similarities • The molecular landscape of aCML is heterogeneous, no one specific alteration, like BCR/ABL for CML • Chromosome changes: 7
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•
•
o Seen in 20–80% of cases o Advancement of sequencing strategies (mate-pair and RNAseq) will help to identify fusion genes, cryptic alterations, and precise rearrangement locations Common mutations include: o SETBP1, ETNK1, ASXL1, N/K-RAS and TET2 o Advancement of sequencing platforms (exome and target capture with greater coverage) will help to uncover additional alterations, including copy number variation o Examining the differential mutational effect in aCML can help us to understand pathobiology/morphologic characteristics: ⁃ Mutant function ⁃ Order of Acquisition ⁃ Allele frequency or copy number of mutation ⁃ Co-expression influence There is no current standard of care o New drugs designed to target mutant gene signaling pathways are a novel and promising therapeutic option o Important to screen individual patients for targetable gene mutations –Emphasis on understanding the pathobiology by using precision genomic approach
Declaration of competing interest The Author of this Review does not have any conflict of interest to disclose. References [1] Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classification of the myeloid neoplasms. Blood 2002;100(7):2292–302. [2] Vardiman J, Hyjek E. World health organization classification, evaluation, and genetics of the myeloproliferative neoplasm variants vol. 2011. Hematology Am Soc Hematol Educ Program; 2011. p. 250–6. [3] Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau MM, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016;127(20):2391–405. [4] Breccia M, Biondo F, Latagliata R, Carmosino I, Mandelli F, Alimena G. Identification of risk factors in atypical chronic myeloid leukemia. Haematologica 2006;91(11):1566–8. [5] Giri S, Pathak R, Martin MG, Bhatt VR. 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Best Practice & Research Clinical Haematology journal homepage: www.elsevier.com/locate/issn/15216926
Atypical CML- the role of morphology and precision genomics Terra Lasho Division of Hematology, Mayo Clinic Rochester, USA
A R T IC LE I N F O
ABS TRA CT
Keywords: Atypical chronic myeloid leukemia Clonal expansion Precision genomics Dysplastic granulopoiesis Clonal evolution
Atypical chronic myeloid leukemia is an esoteric myeloid malignancy with features of both myeloproliferative and myelodysplastic syndromes. This disease is characterized primarily by morphologic-based criteria, and has clinical and molecular features overlapping with other myeloid malignancies. No one molecular abnormality is specific, and multiple mutations are often present in various combinations, due to the malignant multi-step clonal evolution of myeloid malignancies. In this review, we will address what we know about atypical chronic myeloid leukemia; evaluate how the molecular landscape in myeloid malignancies overlaps, and discuss what we can learn by incorporating individualized precision genomic strategies.
Introduction Atypical chronic myeloid leukemia (aCML) is a rare clonal hematopoietic stem cell disorder with absence of a detectable BCRABL1 fusion or Philadelphia (Ph) chromosome. It is currently recognized by the WHO 2016 revised classification as a sub-type of a myelodysplastic/myeloproliferative (MDS/MPN) overlap syndrome, as it displays both proliferative (neutrophil leukocytosis) and dysplastic (prominent granulocyte dysplasia) features [1–3]. The estimated incidence of aCML is 1% (or 1 out of every 100) of Phpositive CML cases with a median overall survival of ~25 months (range, 14–30 months) [4–7]. Clinical presentation is similar to other known myeloid malignancies and includes anemia, splenomegaly, and thrombocytopenia. Patients typically present in the sixth and seventh decade of life and there is a slight male predominance. The normal disease course typically ends in complications from cytopenias, or transformation to acute myeloid leukemia (AML) in 30–40% of cases [4,6,8,9]. The etiology of aCML is not known and the current diagnostic criteria for aCML are a result of a stratification of an accumulation of morphological similarities, which are the most important distinguishing features for diagnosing atypical CML. From what we can tell, the molecular landscape of aCML is heterogeneous. Molecular features include an increased frequency of gene fusions or aneuploid karyotype and/or recurrent somatic mutations affecting genes involved in growth factor signaling, transcriptional regulation, RNA splicing, and epigenetic regulation of DNA methylation/histone modification [6,10,11]. Methods for detecting genetic alterations in aCML have varied as sequencing techniques have rapidly evolved in scope and sensitivity. This along with the paucity of this disease has made molecular profiling difficult. There is no one genetic alteration that is specific to the disease, and in fact, all described genes with pathogenic mutations in aCML have been recognized in related myeloid malignancies. This is conceivable, because along with other myeloid malignancies, aCML contains various combinations of genetic abnormalities due to the malignant multi-step clonal evolution of the disease. The influence of a gene mutation on clinical presentation may not be straightforward and can be due to mutation-specific functional effects, and/or be co-influenced by other mutations, copy number or cellular prevalence of mutations, and the order of mutant acquisition. Ultimately, this challenges the management of, and underscores the marked clinical heterogeneity in these disorders. In this review we will evaluate the clinical parameters in aCML, management, and treatment. We will discuss why there is such
E-mail address: [email protected]. https://doi.org/10.1016/j.beha.2019.101133 Received 2 December 2019; Accepted 3 December 2019 1521-6926/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Terra Lasho, Best Practice & Research Clinical Haematology, https://doi.org/10.1016/j.beha.2019.101133
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clinical variability in myeloid malignancies, and why there is a critical need for a precision genomics-based approach in clinical practice, especially in an esoteric disease like aCML. Precision genomics refers to an individualized approach to understanding molecular pathogenic contributions in disease by taking into account differential influence of varying aspects of genetic alterations. Instead of a “one size fits all” approach, this method provides an individualized mutant genetic/functional summary which has the ability to be translated into a tailored targeted therapy. Background Historically, aCML was considered to be a myeloproliferative subtype of chronic myeloid leukemia (CML) and was first given the name “CML-like syndrome”, because both diseases can present with splenomegaly and display myeloid hyperplasia with left-shifted peripheral granulopoiesis. However, aCML displays some distinct features (primarily no BCR/ABL, poorer survival, and granulocytic dysplasia), and so was subsequently considered an intermediate syndrome somewhere between CML and chronic myelomonocytic leukemia (CMML) on the spectrum of disorders [12]. In 1994, the French-American-British (FAB) Cooperative Leukemia Group studied fifty-one cases of BCR-positive/negative cases and recommended the implementation of measures to distinguish aCML from CML and CMML [13]. These criteria included five quantification parameters (complete white blood cell count [WBC], percentage immature granulocytes, percentage monocytes, percentage basophils, percentage erythroid precursors in bone marrow) and one qualitative parameter (granulocytic dysplasia). During the next couple of years, there were reports supporting the FAB criteria [14], some soliciting the need for better criteria definitions and some claiming that cases satisfied multiple criteria. This raised the question of whether to consider aCML as its own entity, part of a spectrum of CMML, or an unusual evolution of MDS [15–17]. However, in 2001, the WHO created a new disease classification, myelodysplastic (MDS)/myeloproliferative neoplasm (MPN) overlap syndrome. This categorization helped to further differentiate aCML from CML. In 2008, the WHO further distinguished aCML by including a numeric threshold of leukocytosis (≥13 × 109/L) and monocytes (< 10% of WBC), absence of PDGFRA, PDGFRB, and FGFR1 rearrangements, and presence of dysplasia in the granulocytic lineage. In the most recent revision to myeloid malignancy diagnostic criteria, the 2016 WHO revised the criteria for aCML (See Table 1) which included an emphasis on molecular alterations. This includes stratification by presence of SETBP1 and ETNK1 mutations, and if CSF3R mutations are present the case should be rigorously reviewed to exclude alternate diagnosis of chronic neutrophilic leukemia (CNL). (See Fig. 1). Laboratory findings Peripheral blood (PB) WBC counts in aCML are higher than 13 × 109/L (typical range: ~35–96 × 109/L) [4,14]. Immature neutrophils (including promyelocytes, myelocytes, and metamyelocytes) account for ≥10–20% of WBC, but blast percentage is usually less than 5% and always less than 20%. The morphology of neutrophils may be abnormal and predominantly include one of more: hypogranular cytoplasm, exaggerated clumping of nuclear chromatin, and abnormal lobated nuclei (pseudo-pelger huet). Basophilia is usually minimal (≤2%) or absent [4,12,13]. Evidence of dyserythropoiesis is also commonly observed (example is macro-ovalocytosis). However, the most important feature when examining the peripheral blood are the levels of monocytes. This important criterion distinguishes aCML from CMML. In aCML, monocytosis may be present, but is always less than 10% of the WBCs. The bone marrow (BM) in aCML is hypercellular and includes a predominantly granulocytic proliferation. Granulocytic dysplasia is often noticeable with similar features to the PB. Other lineages may be dysplastic as well, with dyserythropoiesis present in up to 50% of cases [4,14]. Staining for CD34 + helps to reveal blasts which may be present, but should not be quantitatively over 20% of all nucleated cells. Although bone marrow histopathology reveals overlap between aCML and CMML which can sometimes be hard to differentiate, dual staining of esterase can distinguish monocytes in the BM biopsy using cytochemical staining and can be helpful for ruling out CMML (See Fig. 2). Overview of clonal evolution of myeloid malignancies Myeloid malignancies arise from an altered hematopoietic stem cell with an acquired influence over the growth and differentiation capacity of one or more cell lineages in the bone marrow and blood. This evolution progresses as a stepwise accumulation of Table 1 WHO Diagnostic Criteria for aCML WHO Diagnostic Criteria (2016) Atypical Chronic Myeloid Leukemia due to increased numbers of neutrophils and their precursors (promyelocytes, myelocytes, metamyelocytes) comprising ≥10% of leukocytes • Leukocytosis which may include abnormal chromatin clumping • Dysgranulopoiesis, bone marrow with granulocytic proliferation and dysplasia, with or without dysplasia in erythroid and megakaryocytic lineages • Hypercellular and bone marrow blast count of less than 20% • Blood of BCR/ABL fusion gene, or rearrangement of PDGFRA and B, FGFR1 or PCM – JAK2 • Absence or minimal absolute monocytosis (usually < 10% of leukocytes) • No • No or minimal basophils (usually < 2%)
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Fig. 1. Morphological stratification for MDS/MPN overlap syndromes. After ruling out a secondary cause, AML, and other fusions, identification for CMML, RARS-T, aCML, and MDS/MPN-U is provided by the corresponding flow chart path. Highlighted in green are several genes which, when observed as mutated are additional indicators of disease type.
Fig. 2. Blood and bone marrow of aCML, BCR-ABL1 negative. A). Wright-giemsa stain (100x) of peripheral blood showing elevated white blood cell count, immature myeloid cells (red arrows) and dysplastic granulocytes (black arrows). B). Wright-giemsa stain (400x) of peripheral showing pseudo-Pelger-Huet cell (black arrow). C). Wright-giemsa stain (200x) of bone marrow aspirate demonstrating granulocytic dysplasia (black arrows). D). Hematoxylin eosin stain (100x) of bone marrow biopsy indicating hypercellularity (involving granulocytic proliferation and dysplasia).
gene mutations which promotes a multi-lineage selective advantage of mutant clonal expansion over normal hematopoiesis. The result is clonal expansion which encourages evolution of disease and consequential complex effects on the genetic, epi-genetic, and microenvironment (See Fig. 3). Appreciating this clonal architecture is essential to understanding how a relatively small group of known gene mutations can orchestrate the extensive clinical and molecular overlap reflected in the proliferative and dysplastic features of myeloid malignancies at diagnosis and throughout the course of disease. A functionally well-characterized gene mutation may influence the transcriptome or epigenome differently in the presence of additional mutations and the type of association as well as the order of mutations have an impact on the kinetics of disease development. We can determine the order of mutant occurrence by assessing mutant variant allele frequencies (VAF) –which is a measure of the ratio of mutant to wild-type allele frequency in a sample, or performing clonogenic assays using BM or PB to expand individual hematopoietic colonies (sub-clones) representing the spectrum of clonal architecture. A 3
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Fig. 3. Clonal evolution influence on disease. aCML is the result of an aberrant clonal expansion. As seen in the top panel, acquisition and propagation of mutations in a cell population promotes expansion and additional mutations which outcompetes normal hematopoiesis. Below is a list of how a disease phenotype can be affected or influenced through clonal evolution.
critical observation in myeloid malignancies is that most of the key genes that are mutated are not specific to a single disease subtype, and in addition, most of the recognized mutated genes can be attributed to ~4 gene function groups: pre-mRNA splicing (SRSF2, U2AF1, ZRSR2 and SF3B1), growth factor signaling transduction (RAS-pathway, CSF3R, JAK2, CALR, MPL, FLT3, KIT, ETNK1), transcription factor (TF)/or TF regulation (RUNX1, CEBPA, GATA2, NPM1), and chromatin and epigenetic regulation of DNA methylation and histone modification (ASXL1, EZH2, BCOR, SETBP1) [10,18]. As the genetic hierarchy of disease is defined, associations can be easier to predict regarding clinical and genetic classifications. We have learned a lot about this from several large scale single-cell based analyses and clonal reconstruction datasets, involving acute myeloid leukemia (AML) [19–22]. One important observation is that mutations involving the genes DNMT3A, ASXL1, and TET2, are not only important prognostic indicators of disease, they have been shown to appear years before any sign of disease, and are considered CHIP genes “clonal hematopoiesis of indeterminate potential” because of their prevalence in healthy people > 50 years of age. The incidence of acquiring mutations in these genes rises with age, and they are associated with risk of development of a blood disorder [23]. Mutations in CHIP genes persist in cases of relapse and remissions, and their presence have been shown to provide a selective multi-lineage advantage to mutant over wild-type hematopoietic progenitor cells [24–26]. Because of this, they are considered “pre-leukemic” and important for disease initiation. CHIP mutations are almost always associated with other recurrent mutations, suggesting functional cooperation in acquisition/progression of leukemia as observed in AML cohorts as well as in other myeloid malignancies. However, their existence is not critical for disease [27,28]. CHIP genes may or not present as CHIP and are gene members of one of most recognized mutated gene functional categories: chromatin and epigenetic regulation. Almost 90% of de novo AMLs with a normal karyotype follow a classical hierarchy of acquisition of mutations, which involves a successive occurrence of a mutation in epigenetic regulation including DNA methylation, followed by a mutation in a TF, then finally, a mutation involving signaling/tyrosine kinase pathway [19,20,22,29]. In a study of one thousand five hundred and forty AML cases, the order of mutations (identified by using variant allele frequencies) revealed CHIP mutations first (TET2 [16%] and DNMT3A [54%]), followed by TF mutations (NPM1), and finally signaling mutations (FLT3 [39%], NRAS/PTPN11 [34%]). A second clonal hierarchy has been described in de novo AML which excludes CHIP mutations, but keeps the TF (RUNX1, GATA2, CEBPA) and then signaling gene acquisition order [21]. This is important because it provides a kind of blueprint toward the evolution of a myeloid malignancy, that is, epigenetic changes which promote or at least provide a background for transcriptional/signaling dysregulation, affecting differentiation, proliferation, and survival of cell types. Interestingly, in secondary AML, which frequently arises from MDS (in more than 20% of cases), the acquisition of mutations differs, and instead involves splicing genes (SRSF2, SF3B1, U2AF1, and ZRSR2) and chromatin modifiers (BCOR, ASXL1, EZH2). In a study of one hundred ninety-four cases of either secondary or therapy-related AMLs, the two most frequent mutated genes involved ASXL1 (32%) and SRSF2 (20%) [20]. Sixty-three percent of SRSF2-mutated s-AML are also mutated with ASXL1. This is significant because ASXL1 mutations are CHIP-related, and patients with MDS are of advanced age, so it is logical that they are present. Splicing mutations appear to be associated with the pathogenesis of myelodysplasia as in a study of nine hundred and forty-four cases of MDS they represent the most mutated functional gene group (mutated in 64%) [30]. The six genes mutated in more than 10% 4
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of MDS cases included: TET2, ASXL1, SRSF2, SF3B1, DNMT3A, and RUNX1. In several other smaller studies, the most recurrent mutant gene associations in MDS were between IDH2, STAG2, ASXL1, RUNX1, and BCOR, with ASXL1 being the most frequent [31–33]. TET2 mutations were associated with SRSF2 mutations, and this is very significant for CMML also, where the thought of early clonal dominance of TET2 mutated cells have the ability to skew myeloid differentiation toward the granulomonocytic lineages (possibly elucidating the CMML/monocytosis relationship) [34]. Overall, VAF analysis in MDS demonstrated that mutations in DNA methylation and splicing appear before all others and mutations in signaling pathways occurred last, and this has been verified in multiple studies [31,32,35,36]. Finally, in BCR-ABL-negative MPNs which have evolved to blast phase, (1% in essential thrombocythemia [ET], 7% for polycythemia vera [PV], and 20% in primary myelofibrosis [PMF]), mutations up-regulating the JAK/STAT-signaling pathway (CALR, and MPL) appear first followed by mutations in chromatin modifiers (TET2, ASXL1 or DNMT3A). In PMF, JAK2 mutations appear first followed by a mutation in chromatin modifiers (ASXL1 or EZH2). However, in ET, mutations in JAK2 occur as a sub-clonal event to TET2, DNMT3A, or SF3B1 [37,38]. This demonstrates that the order of mutations is important to identify as this can influence the differential diagnostic criteria for the development of disease (in this case, PMF vs. ET, which includes the ratio of granulocyte and erythroid expansion and atypical features of megakaryocytes). Finally, the allele frequency of a JAK2V617F mutation can change (increase in VAF), and this can influence the disease enough to change the diagnosis from ET to PV [39]. Whether this is due to the increase in clonal burden of JAK2V617F due to VAF or gene dosage, or a result of pre-existing or additional somatic mutations, reinforces the need to completely characterize the mutational environment in order to have an effective perspective of molecular pathogenesis. Overall, it is critical to understand that not only the function of a mutation, but the order of acquisition, VAF, cellular presence (gene dosage), or co-expression with other mutations can influence the pathophysiology and disease clinical presentation in myeloid malignancies. Unlike CML, which is clearly defined by the consistent presence of BCR-ABL1 fusion, we have a limited understanding of aCML pathobiology because of the lack of specific molecular markers and overlapping morphologic features with other MDS/MPN syndromes, which also include: MDS/MPN-U and CMML. According to the International MDS/MPN Working Group, although “there have been a number of contributions to literature, the MDS/MPN overlap syndromes have considerable molecular heterogeneity, and lack of specific markers” [40]. Just recently, the largest study based on genetic-assessment of MDS/MPN overlap syndromes was published. In this study, whole exome sequencing was performed on one hundred and fifty-eight patients which included MDS/MPN overlap syndromes (including n = 27 aCML) [41]. Unlike previous studies where disease sub-types were initially stratified by WHO criteria and then individually screened and compared for molecular alterations, this group performed an unsupervised mutational screening to then stratify disease types by mutation profile. The overall findings revealed a complex overlap among genetic mutations and disease. No single mutation or combination of mutations defined a singular disease sub-type. In essence, the findings highlight the idea that dose and sequential effect of clonal evolution, and the order of occurrence contribute to the heterogeneity of the clinical features within this group of diseases. This helps to explain why there is such extreme difficulty in clarifying diagnostic classifications based solely on histology for some cases. What do we do with this information? Well, (in addition to understanding the mutational co-influence perspective from the AML studies above) this report did reveal some interesting mutational relationships which correlated to clinical features. They reported that more than half of the patients demonstrated co-occurring mutations in at least three different gene functional pathways. And in the majority of cases, by examining the VAF structure, they provide evidence that patients had at least two mutations involving epigenetic regulation and splicing factors and these were in the dominant clone, with acquisition of additional mutations. We know that splicing mutations are frequent in MDS. They also observed that mutations in ASXL1, TET2, and EZH2 correlated with older age (consistent with what we discussed above as ASXL1 and TET2 are CHIP-related genes). Mutations in CSF3R, which is a cytokine that controls the regulation of granulocytes, were (of course) associated with high neutrophil count, but also presence of splenomegaly and low monocyte count (CSF3RT618I mutations have been reported as highly associated to CNL [42]). The presence of signaling mutations, NRAS in particular, were associated with low hemoglobin, platelet count, and neutrophil percentage, and high monocyte percentage. ASXL1 mutations were associated with high WBC and need for platelet transfusion. TET2 mutations were associated with low neutrophil percentage, high monocyte percentage, BM dysplasia, low platelet count, and consistently high platelet transfusion requirement. And finally, SETBP1 mutations were associated with high hemoglobin level and high platelet count. In addition to singular mutations, aberrant karyotypes and copy number variations were also detected in these cases. Molecular landscape of aCML For aCML, the most frequent mutations described (to varying degrees) have been found in SETBP1, ASXL1, (N/K)RAS, SRSF2, TET2, CBL, CSF3R, and ETNK1 [6]. [10,11,18,40,43–47] However, the comparison of longitudinal studies is limited to the range and depth of the technique applied (See Table 2). Of these, mutations in SETBP1 and ETNK1 appear to be the most recurrent genes mutated in aCML and are seen in up to a third of patients [45]. [48,49] Mutations in SETBP1 in fact have been reported to have a seven-fold lower incidence in CMML and uncommon occurrence in MPN, suggestive of this as a potential molecular marker a useful diagnostic tool to differentiate aCML between these clinically similar entities (and represented as a mutation of diagnostic emphasis for aCML by WHO-2016 revised criteria) [45,49,50]. Multiple studies have also shown that mutated SETBP1 is associated with a more adverse clinical presentation (higher leukocyte count, lower hemoglobin level, thrombocytopenia), suggesting this mutation is not only relevant to leukemic oncogenesis, but also provides important prognostic value [45,49,51]. There is also a strong correlation of mutated SETBP1 with mutations involving the chromatin remodeling gene, ASXL1, a 5
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Table 2 Mutational Gene Spectrum in aCML Type
Gene
Name
Frequency (%)
Reference
Epigenetic regulation of DNA methylation/ histone modification
IDH1/IDH2
Isocitrate Dehydrogenase 1 (cytosolic), Isocitrate Dehydrogenase 2 (Mitochondrial) Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit Additional Sex Comb Like 1 SET Binding Protein 1 Tet Methylcytosine Dioxygenase 2 Serine And Arginine Rich Splicing Factor 2 U2 Small Nuclear RNA Auxillary Factor 1 Splicing Factor 3b Subunit 1 Zinc Finger CCCH-Type, RNA Binding Motif And Serine/ Arginine Rich 2 N/K Proto-Oncogene Receptor, GTPase Janus Kinase 2 Colony Stimulating Factor 3 Receptor (Granulocyte) Calreticulin MPL Proto-Oncogene, Thrombopoietin Receptor KIT Proto-Oncogene Receptor Tyrosine Kinase Fms Related Tyrosine Kinase 3 Fms Related Tyrosine Kinase 3 - internal tandem duplication Cbl Proto-Oncogene Ethanolamine Kinase 1 Protein Tyrosine Phosphatase, Non-Receptor Type 11 Runt Related Transcription Factor 1 CCAAT Enhancer Binding Protein α Nucleophosmin 1 IKAROS Family Zinc Finger 1
0 to 4
[6,52]
8 to 20
[48,52]
20 to 66 12 to 33 16 to 41 12 to 40 0 to 20 8 4
[6,49,52] [6,11,45,48,52] [11,52] [11,52] [48,52] [52] [52]
8 to 3 to 0 to 0 to 0 to 0 7.1 4
[6,9,48,52] [6,11,52] [6,10,11,52] [6,11,47,52] [6,11,52] [6] [6] [52]
EZH2 ASXL1 SETBP1 TET2 SRSF2 U2AF1 SF3B1 ZRSR2
RNA splicing
Signal Transduction
(N/K) RAS JAK2 CSF3R CALR MPL KIT FLT3 FLT3-ITD CBL ETNK1 PTPN11 RUNX1 CEBPA NPM1 IKZF
Transcription Factor
35 8 40 4 2
0 to 10 8 to 8.8 4 12 11.8 0 0
[11,48,52] [46,52] [52] [52] [6,52] [6,52] [52]
commonly mutated oncogene with detrimental prognostic impact, suggesting their potential cooperative function in leukemic progression of aCML. In a cohort of 39 cases, co-mutated SETBP1 and ASXL1 occurred in 48% of aCML patients. ASXL1 has already been shown to be a high risk prognostic marker in AML, MDS, and PMF, and may also provide further prognostic value in aCML [11,49,53]. However, mutations in ASXL1 were not shown to affect overall survival in a study by Patnaik et al. [52]. In 2015, recurrent mutations in ETNK1 were found at a prevalence of 8.8% in sixty-eight cases of aCML. All mutations were heterozygous, and in two cases were present concurrent with SETBP1 mutations [48]. The ETNK1 gene protein functions as a kinase in phospholipid biogenesis. The exact functional role of mutations in this gene is not yet clear, however, mutations appear to be relatively specific to aCML, CMML, and systemic mastocytosis with associated eosinophilia [48,54]. Although cases of aCML have myeloproliferative features, including splenomegaly and leukocytosis, mutations in the most wellknown driver genes found in myeloproliferative disorders (JAK2, MPL, and CALR) are typically not found in aCML. However, JAK2V617F has been cited with low frequency (~4–8%) [6,18,55]. This is an important observation suggesting that this type of mutation is unlikely to be a driver mutation in aCML, but is still valuable in differentiating the MDS/MPN overlap syndromes from MPN [56,57].
Cytogenetic Karyotypic abnormalities in aCML range widely and have been reported in 20–80% of patients. The most common abnormalities are +8 and del (20q), but abnormalities of chromosomes 13, 14, 17, 19 and 12 are also commonly reported [9]. There was a report of Trisomy 14 in a case of aCML in 1990 [58]. Several case studies resulting in fusions have also been reported [59–62]. Sequencing detection methods are now becoming available, including RNAseq, which can interrogate the transcriptome and detect the presence Table 3 Other Genetic Alterations in aCML Type
Chromosomal abnormality
Most common
+8 and del (20q) abnormalities of chromosomes 13, 14, 17, 19 and 12 Trisomy 14 46XX,t(2; 13; 2;21)(p13; q12; q33; q11.2) t(3; 21)(q22; q22) t(1; 9)(p34; q34) t(7; 11) (p15; p15)
Case Case Case Case Case
Report Report Report Report Report
Fusion Gene
6
Reference [9]
SPTBN1-FLT3 RYK/ATP5O CSF3R/SPTAN1 NUP98-HOXA9
[58] [59] [60] [61] [62]
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of aberrant fusion genes. Therefore, the presence/identification of fusion genes may become more important and specific as more cases are probed (See Table 3). Prognostic factors and treatment Atypical CML has an aggressive disease course, with a poor prognosis and a median survival of ~25 months (range, 14–30 months) [4–6]. Clinical prognostic factors included age > 65 years, female, hemoglobin < 10 g/dL, WBC count > 50 × 109/L, and immature circulating precursors. In addition, mutations in ASXL1 and SETBP1 have been associated with high risk [63]. No current standard of care exists for aCML. Because of the short predicted overall survival of the disease, there is no prognostic algorithm advising aggressiveness of treatment. Constitutive symptoms as well as anemia, thrombocytopenia, progressive leukocytosis, and symptomatic splenomegaly should be managed if present with standard supportive care measures used currently for MDS and MPN. Allogeneic stem cell transplant appears to be the only long-term treatment option; however, most patients are not eligible due to advanced age at diagnosis and comorbidities. Additional current treatment options include hypomethylating agents, which have demonstrated transient improvement in clinical symptoms [7,64]. Rarely, complete and partial responses have been reported with hydroxyurea, although short lived [8,65,66]. Hydroxyurea can also be used for supportive care in the presence of another treatment to control progressive leukocytosis or splenomegaly. Standard IFN-α therapy has been described with varying durability of response [8,65,66]. In a phase 2 study of PEFIFN-a-2b, two aCML patients out of five achieved a complete remission after only 3 months of therapy. Both patients were discontinued due to toxicity; however, the median duration of therapy was 36 and 38 months. This type of therapy will need to be further assessed [67]. However, the most interesting and promising therapeutic options include targeted therapies currently available for specific mutational profiles. This approach will be based on the ability to utilize an individualized precision-genomic-based approach to assess genetic/functional profiles in aCML (and related myeloid malignancies). Therefore, for practicing clinicians now, it is essential to molecularly screen incoming patients using available myeloid mutation genotyping panels not only to better understand the contribution of these mutations to the pathobiology, but to help to identify targets for individualized gene-targeted therapy [64,68–74]. Summary and future directions Atypical CML is heterogeneous, both clinically and molecularly, to other myeloid malignancies, especially MDS/MPN overlap syndromes. This is partly due to the difficulty in comparing reports from a longitudinal characterization over several decades of redefined diagnostic criteria/emerging sequencing techniques. However, the mutated gene profile in aCML is similar to other MDS/ MPN overlap syndromes and additional myeloid malignancies. Large scale clonality studies have demonstrated that the function of a specific mutation may be less important than its order of acquisition, allele frequency or copy number, and/or co-influence with other mutations, in determining the pathobiology of a disease. This is well described in large AML single-cell and clonal studies, where the order of acquisition of types of mutation relates to disease clinical presentation and progression. These studies have also verified that splicing mutations are associated to myelodysplasia, and early acquisition of TET2/SRSF2 mutations influences the granulomonocytic predisposition in CMML and may drive the characteristic feature of monocytosis that distinguishes CMML from other MDS/MPNs. Precision genomics is a paradigm changing method to individually approach disease on a case by case basis, by taking into account differential influence of varying aspects of genetic alterations. Instead of a “one size fits all” approach, this method provides an individualized mutant genetic/functional summary which has the ability to detect early and distinct phases of disease, and be incorporated into a tailored targeted therapy. This ability has already demonstrated promising strategies in aCML, including the recent proposal of considering the reactivation of PP2A as a therapeutic strategy in SETBP1-mutated cells [75]. Practice points
• There is a current lack of understanding of the molecular landscape in aCML. • Practicing clinicians must utilize available myeloid screening panels to interrogate cases of aCML –This will help identify targets for individualized therapy • Push to incorporate Precision Genomics into practice: o o o o
Paradigm change from “one-size-fits-all” to individualized fingerprint as a diagnostic strategy Individualized case assessment by merging deep screening techniques Ability to detect early and distinct phases of disease Pharmacogenomics -provide individualized targeted therapy “personalized medical cocktail”
Research agenda
• Etiology of aCML is not well understood • Diagnostic criteria for aCML are a result of a stratification of an accumulation of morphological similarities • The molecular landscape of aCML is heterogeneous, no one specific alteration, like BCR/ABL for CML • Chromosome changes: 7
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•
•
o Seen in 20–80% of cases o Advancement of sequencing strategies (mate-pair and RNAseq) will help to identify fusion genes, cryptic alterations, and precise rearrangement locations Common mutations include: o SETBP1, ETNK1, ASXL1, N/K-RAS and TET2 o Advancement of sequencing platforms (exome and target capture with greater coverage) will help to uncover additional alterations, including copy number variation o Examining the differential mutational effect in aCML can help us to understand pathobiology/morphologic characteristics: ⁃ Mutant function ⁃ Order of Acquisition ⁃ Allele frequency or copy number of mutation ⁃ Co-expression influence There is no current standard of care o New drugs designed to target mutant gene signaling pathways are a novel and promising therapeutic option o Important to screen individual patients for targetable gene mutations –Emphasis on understanding the pathobiology by using precision genomic approach
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