Molecular Genetic Profile of Myelofibrosis: Implications in the Diagnosis, Prognosis, and Treatment Advancements

Abstract

Myelofibrosis (MF) is an essential element of primary myelofibrosis, whereas secondary MF may develop in the advanced stages of other myeloid neoplasms, especially polycythemia vera and essential thrombocythemia. Over the last two decades, advances in molecular diagnostic techniques, particularly the integration of next-generation sequencing in clinical laboratories, have revolutionized the diagnosis, classification, and clinical decision making of myelofibrosis. Driver mutations involving JAK2, CALR, and MPL induce hyperactivity in the JAK-STAT signaling pathway, which plays a central role in cell survival and proliferation. Approximately 80% of myelofibrosis cases harbor additional mutations, frequently in the genes responsible for epigenetic regulation and RNA splicing. Detecting these mutations is crucial for diagnosing myeloproliferative neoplasms (MPNs), especially in cases where no mutations are present in the three driver genes (triple-negative MPNs). While fibrosis in the bone marrow results from the disturbance of inflammatory cytokines, it is fundamentally associated with mutation-driven hematopoiesis. The mutation profile and order of acquiring diverse mutations influence the MPN phenotype. Mutation profiling reveals clonal diversity in MF, offering insights into the clonal evolution of neoplastic progression. Prognostic prediction plays a pivotal role in guiding the treatment of myelofibrosis. Mutation profiles and cytogenetic abnormalities have been integrated into advanced prognostic scoring systems and personalized risk stratification for MF. Presently, JAK inhibitors are part of the standard of care for MF, with newer generations developed for enhanced efficacy and reduced adverse effects. However, only a minority of patients have achieved a significant molecular-level response. Clinical trials exploring innovative approaches, such as combining hypomethylation agents that target epigenetic regulators, drugs proven effective in myelodysplastic syndrome, or immune and inflammatory modulators with JAK inhibitors, have demonstrated promising results. These combinations may be more effective in patients with high-risk mutations and complex mutation profiles. Expanding mutation profiling studies with more sensitive and specific molecular methods, as well as sequencing a broader spectrum of genes in clinical patients, may reveal molecular mechanisms in cases currently lacking detectable driver mutations, provide a better understanding of the association between genetic alterations and clinical phenotypes, and offer valuable information to advance personalized treatment protocols to improve long-term survival and eradicate mutant clones with the hope of curing MF.

Keywords: JAK inhibitor; epigenetic regulation; molecular diagnostics; mutations; myelofibrosis; myeloproliferative neoplasm; next-generation sequencing; primary myelofibrosis.

Figure 3

Schematic diagram of the clonal evolution patterns in myelofibrosis (MF). Four patterns of clonal evolution are illustrated based on published clinical and experimental studies. Clones/subclones harboring different mutation(s) are represented in different colors, indicated in the lower left corner of each panel. Most clinical myeloproliferative neoplasm (MPN) cases are likely diagnosed in steps 2–4. More complex and combination patterns exist. The diagram was created using Pyfish 1.0.3 [70] (https://pypi.org/project/pyfish/ (accessed on 6 December 2023)). “Driver” is a driver mutation of myeloproliferative neoplasm (MPN) in one of the three genes: JAK2, CALR, or MPL. (A) Linear evolution pattern. Driver mutation emerges from the normal hematopoietic cell population; additional mutations (Mut1, Mut2) acquired stepwise in the same clone with driver mutations. An additional mutation (Mut2) may drive proliferation and expansion of the subclone to become the major neoplastic population. (B) Driver mutation acquired in a cell with pre-existing mutation(s). MPN driver mutation acquired in a cell of clonal hematopoiesis of indeterminate significance (CHIP), with mutation(s) represented as D/A/T (DNMT3A, ASXL1, TET2) mut. In addition to the driver mutation, other mutations (Mut2, Mut3) may also be acquired later. (C) Branching subclonal evolution pattern. Within a clone with a driver mutation, multiple subclones (driver+Mut1 and driver+Mut0) may coexist, and some may acquire other mutations (Mut2, Mut3) sequentially; after acquiring Mut3, the clone gains a proliferation advantage, driving disease progression or transformation to acute myeloid leukemia (AML). (D) Paralleled subclonal evolution. An MPN clone (Driver) and a clone with no MPN driver mutation (Mut0) coexist, multiple subclones may develop from the clone independent of the MPN driver clone, and some (illustrated as Mut0+Mut2+Mut3 clone) may gain a proliferation advantage, becoming the major clone with disease progression or transformation to AML.

Figure 1

Myelofibrosis (MF, case and images by L.Z.). Bone marrow biopsy images are from a 64-year-old woman diagnosed with essential thrombocythemia (ET) 15 years ago and on intermittent hydroxyurea therapy. (A) The hypercellular bone marrow shows frequent atypical megakaryocytes, some displaying hyperchromatic nuclei (green arrows) and forming clusters (black arrows) (H&E stain, 100×, scale: 100 μm). (B) Reticulin stain (200×, scale: 50 μm) reveals moderate myelofibrosis (MF grade 2 of 3, representative areas with increased reticulin fiber forming meshwork are indicated by black arrows). Next-generation sequencing of 75 genes associated with myeloid neoplasms revealed JAK2 V617F at 34.5% and DNMT3A R635W at 18.9%. The difference in the variant allele frequency suggests that either the DNMT3A mutation is subclonal or the JAK2 mutation is homozygous. At this stage, the morphologic features and mutation profile of post-ET MF are indistinguishable from those of primary myelofibrosis (PMF).

Figure 2

Number of mutations in each sample, essential thrombocythemia and polycythemia vera (ET and PV) versus primary myelofibrosis (PMF). Data source: The AACR GENIE public database [32] (see text for the link to the dataset). ET and PV: 492 samples; PMF: 227 samples. The bar height is displayed as the percentage of samples in each category (Y-axis), and the absolute number of samples in each category is displayed on top of the bar. There is a significantly higher percentage of PMF cases harboring >2 mutations compared with ET and PV cases (49.78% vs. 20.73%, p < 0.00001 by Fisher exact test).