Molecular profile of patients with myelofibrosis: a 10-year experience

ABSTRACT Objective To analyze the karyotype test and myeloid panel with next-generation sequencing findings in patients with myelofibrosis, and to compare transplant characteristics in patients referred for bone marrow transplantation. Methods Retrospective, single-center study with patients diagnosed with myelofibrosis treated at Hospital Israelita Albert Einstein between 2010 and 2020. Results A total of 104 patients with myelofibrosis were examined. Patients who had not been submitted to tests in our service were excluded. The final sample comprised 69 patients. Of these 69, 56 were submitted to karyotyping and 22 to myeloid panel with next-generation sequencing. Karyotype was normal in 60% of the patients and altered in 40%. The prevalence of high-risk molecular mutations was higher in patients referred for bone marrow transplantation (100% versus 50%). The median follow-up of transplant patients was 2.4 years and the overall survival at 2 years was 80% (95%CI: 62-100%). Conclusion The molecular analysis enables estimating the patient’s risk and thus instituting more aggressive treatment such as bone marrow transplant for patients at higher risk, being a relevant tool to guide therapy. Given the significance of molecular analysis for therapeutic decision-making in myelofibrosis, collection and disclosure of data on the prevalence of cytogenetic changes and findings of next-generation sequencing in affected patients is important.

marrow transplant: 100% versus 50%. The molecular analysis enabled estimating the patient's risk and is a relevant tool to guide therapy.

Molecular profile of patients with myelofibrosis: a 10-year experience
❚ INTRODUCTION Myelofibrosis (MF) is a BCR-ABL-1 negative myeloproliferative neoplasm. This type of neoplasm can be primary ("de novo" presentation) or secondary to polycythemia vera (PV) or essential thrombocythemia (ET). (1,2) Primary MF is more prevalent and affects 4 to 6 people per 100,000 population, whereas post-ET and post PV MF affect 0.5 to 1.1 and 0.3 to 0.7 per 100,000 people respectively. (3,4) Myelofibrosis is slightly more common in elderly males with median age at diagnosis of 64 years. (5) The estimated median overall survival of patients with primary MF or MF secondary to PV is 4.5 years, compared to 7.06 years in patients with MF secondary to ET. (1) The primary causes of death include leukemic transformation, cardiovascular events and complications of cytopenia, such as infection or bleeding. (2) Myelofibrosis is associated with the presence of 3 cardinal and often mutually exclusive mutations: janus kinase 2 V617F (JAK2V617F), calreticulin einstein (São Paulo). 2023;21:1-5 (CALR) and myeloproliferative leukemia virus (MPL) oncogene. (2) Only 10% of patients are triple negative (no JAK2, CALR or MPL mutations). These patients have lower survival rates and higher risk of progression to acute myeloid leukemia (AML). (1) Genetic markers are determinant of outcomes in patients with MF and have been incorporated into formal prognostic systems, such as MIPSS70+ and GIPSS. (1) Other risk factors which contribute to progression to AML include unfavorable karyotypes, circulating blast percentages higher than 3%, platelet counts less than 50,000, TP53, and highrisk somatic mutations such as ASXL1 (frequency of 22%), SRSF2 (9%), EZH2 (5%), IDH1/2 (3%) and U2AF1 Q157 (16%). (1) Despite the availability of new therapeutic agents to tackle MF, the only treatment with curative potential is allogeneic bone marrow transplantation (BMT). (2) Unfortunately, BMT is associated with at least 50% of transplant-related deaths or severe morbidity such as graft-versus-host disease (GVHD) in MF patients. (2) In a Mayo Clinic study with MF patients submitted to allogeneic BMT, the 5-year overall survival was 62%. (6) Hence, risks associated with BMT must be weighed according to life expectancy, should the patient not receive BMT. For this type of assessment, molecular genetic risk factors of patients must be determined. (2) ❚ OBJECTIVE To analyze the karyotype test and myeloid panel with next-generation sequencing findings in patients with myelofibrosis, and to compare transplant characteristics in patients referred for bone marrow transplantation.

❚ METHODS Study design
Retrospective, single-center study based on medical records of patients diagnosed with MF treated at Hospital Israelita Albert Einstein (HIAE) between 2010 and 2020. Patient data (age, sex, clinical status, date of diagnosis, karyotype, mutations, treatments, last follow-up, and death) were collected, as well as transplant data of patients undergoing BMT (conditioning regimen, cell source, type of transplant, neutrophil engraftment, and occurrence of GVHD).

Inclusion criteria
Patients diagnosed with myelofibrosis treated at HIAE between 2010 and 2020. Primary and secondary MF diagnosis were defined according to 2016 World Health Organization (WHO) diagnostic criteria. (1) Patients with available clinical, laboratory and therapeutic data in medical records.

Exclusion criteria
Inability to retrospectively collect patient and clinical outcome data.

Definitions and outcomes
Overall survival was defined as survival from the date of diagnosis to the date of death from any cause. Patients who were alive at the time of analysis were censored.

Statistical analysis
Overall survival was estimated using the Kaplan-Meier method. Categorical and continuous variables were reported using descriptive statistics. Statistical tests were performed using R software, version 4.0.0.
The prevalence of high-risk somatic mutations in patients submitted to NGS in this sample was higher than the prevalence reported in the general population ( Table 2). (1) This may have reflected that, in this study, NGS panels were used to help decide whether patients at higher risk should be referred for BMT.    Most drugs available for MF treatment are palliative and aimed to alleviate symptoms, reduce complications and improve quality of life, with no impact on the natural history of disease or survival. (2) The only potentially curative treatment is BMT, which has a high morbidity and mortality. (3) In spite of the small number of patients undergoing BMT in this study, results were consistent with findings reported in prior studies using the BuFlu regimen with appropriate busulfan doses. Optimization of conditioning regimens and use of JAK2 inhibitors are thought to improve engraftment rates in BMT for myelofibrosis. However, new strategies are needed to reduce GVHD incidence and post-transplantation relapse rates, to improve clinical outcomes of transplanted patients.
Gowin et al. compared the survival of 1,928 patients with myelofibrosis submitted to allogeneic BMT (551) or clinical treatment (1,377). (7) Patients undergoing allogeneic transplantation had shorter 1-year survival relative to patients receiving clinical treatment. However, over the course of 6-year follow-up, MF patients treated with BMT who had intermediate-1 or higherrisk DIPSS had better long-term survival, despite higher early mortality rates. (7) Low-risk patients did not benefit from BMT and had poorer survival rates compared to patients undergoing clinical treatment.

❚ CONCLUSION
Optimization of conditioning regimens and use of JAK2 inhibitors are thought to improve engraftment rates in bone marrow transplantation for myelofibrosis. Ruxolitinib has important immunosuppressive effects, and may help control graft-versus-host disease after allogeneic bone marrow transplantation. However, new strategies are needed to reduce the incidence of graft-versus-host disease and improve clinical outcome of these transplanted patients. Karyotype analysis and graft-versus-host disease can be used to estimate a patient's risk. These approaches may inform the need of more aggressive treatment in higher risk patients, such as bone marrow transplantation. Molecular analysis is thought to be a relevant tool to guide therapy.