Genetic alterations in myeloid sarcoma among acute myeloid leukemia patients: insights from 37 cohort studies and a meta-analysis

Introduction Variations in mutation rates among acute myeloid leukemia (AML) patients with myeloid sarcoma (MS) underscore the need for a thorough examination. This meta-analysis was conducted to fill the information gap concerning mutation frequencies in AML patients presenting with MS. Materials and methods This study included retrospective and prospective cohorts. It examined genetic alterations in AML patients with and without MS across all age groups. The search strategy employed terms such as “acute myeloid leukemia,” “extramedullary,” “granulocytic sarcoma,” “myeloid sarcoma,” and “leukemic cutis” in the EMBASE, MEDLINE, and Scopus databases. Excluded from the study were reviews, case reports, and case series with fewer than 10 cases. Statistical analyses were performed with Review Manager 5.4 software. Results The primary analysis incorporated data from 37 cohorts involving 5646 diagnosed AML patients and revealed a 17.42% incidence of MS. The most prevalent mutation among AML patients with MS was FLT3-ITD, with a pooled prevalence of 17.50% (95% CI 12.60% to 22.50%; I2 82.48%). The dominant fusion gene was RUNX1::RUNX1T1, displaying a pooled prevalence of 28.10% (95% CI 15.10% to 41.20%; I2 96.39%). In comparison, no significant intergroup differences were observed for NPM1, FLT3-ITD, KIT, and IDH2 mutations. Interestingly, the CEBPA mutation exhibited protective effects for MS patients, with an odds ratio of 0.51 (95% CI 0.32 to 0.81; I2 0%). Conversely, the NRAS mutation was associated with an increased risk of MS development, with an odds ratio of 5.07 (95% CI 1.87 to 13.73; I2 0%). Conclusion This meta-analysis sheds light on the prevalence of genetic mutations in AML patients with MS, providing insights into the unique characteristics of the mutations and their frequencies. These discoveries are crucial in informing therapeutic and prognostic decisions for individuals with myeloid sarcoma.


Introduction
Acute myeloid leukemia (AML) is characterized by the uncontrolled proliferation of myeloid stem cells and impaired differentiation (1).In 2019, the United States observed an estimated total of more than 20 000 AML cases, with certain studies suggesting an age-adjusted incidence rate of 3.43 cases per 100 000 individuals annually (2).Extensive research into AML pathogenesis has identified numerous mutations and cytogenetic abnormalities as pivotal contributors to disease onset (3,4).In 2016, the World Health Organization classified myeloid sarcoma (MS) as an AML subtype.This classification was retained in the World Health Organization's updated 2022 classification and the 2022 International Consensus Classification, wherein MS remains a recognized entity (5)(6)(7).
MS is a tumor mass formed of myeloblasts outside the bone marrow (3).Predominantly, MS affects patients diagnosed with AML or chronic myeloid leukemia, constituting approximately 9% of these cases (8).Notably, the prevalence of MS is greater in males than females, and the condition predominantly affects individuals aged 46 to 59 years (3,8,9).The pathophysiology of MS, especially the migration of cells to extramedullary sites, remains elusive.Prevailing hypotheses suggest that the development of MS may be linked to leukemic cells expressing CD56 (neural cell adhesion molecule) (4).These cells possibly bind to tissues commonly associated with MS manifestations (3).
MS commonly manifests in extramedullary sites such as the skin, bones, soft tissues, and gall bladder (4).However, some studies also document its occurrence in rarer locations, including the pleura, penis, and vulva (10)(11)(12).The prognosis for MS patients tends to be unfavorable and can vary based on the location of the lesion and its molecular attributes (1,8,13).In modern diagnostic methodologies, next-generation sequencing (NGS) has emerged as a crucial tool for identifying mutations in AML patients, including those with MS (3).The NPM1 mutation is the most common mutation found in MS; other common mutations and fusion genes include KRAS, NRAS, KIT, CEBPA, IDH1, IDH2, RUNX1:: RUNX1T1, and CBFB::MYH11 (3, 10, 14-16).However, variations persist in the reported incidence of each mutation in MS among studies (12,14,16).Moreover, a previous report indicated variations in the prevalence of chromosomal abnormalities and/or molecular mutations among different countries (17).However, there is currently no available data regarding these variations specifically within the subgroup of AML with MS.
Consequently, this systematic review and meta-analysis compiled and analyzed data on the incidence of each mutation from all pertinent sources.Our objective was to better understand the specific characteristics and precise prevalence of genetic mutations in AML patients presenting with MS.

Data sources and searches
Six researchers (S.U., K.K., S.P., T.K., W.O., and T.R.) independently searched for articles published within the EMBASE, MEDLINE, and Scopus databases from their inception up to August 1, 2023.The search terms included "acute myeloid leukemia," "extramedullary," "granulocytic sarcoma," "myeloid sarcoma," and "leukemic cutis."A comprehensive description of the search strategy is provided in Supplementary Data 1.Our systematic review and meta-analysis strictly followed the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines, as elaborated in Supplementary Data 2.

Selection criteria and data extraction
Studies included in the meta-analysis were retrospective or prospective cohort studies on AML with MS and with a primary outcome aligned with our research objective.We excluded reviews, case reports, and case series with fewer than 10 cases.The primary objective of this analysis was to determine the incidence of each mutation in AML patients with MS, while the secondary aim was to compare the mutational statuses of AML patients with and without MS.To ascertain study eligibility, four researchers (S.U., K.K., S.P., and T.K.) independently assessed the titles and abstracts of the retrieved studies.They also reviewed the references in the selected studies to identify any additional pertinent research.In instances of disagreement about the inclusion of specific studies, consensus was reached through mediation with two other investigators (W.O. and T.R.).

Quality assessment
The quality assessment of the included studies was independently conducted by two researchers (S.U. and K.K.) using the Newcastle-Ottawa quality assessment scale (18).

Statistical analysis
We analyzed the data with Review Manager 5.4 software provided by the Cochrane Collaboration (London, United Kingdom).The inverse variance method was employed to compute pooled odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) for each gene across the studies (19).The prevalence of the genetic alterations was meta-analyzed and pooled using the binary random-effects model using the DerSimonian-Laird method (Open Meta-Analyst for Windows 8) (20).Given the anticipated variability among the incorporated studies, a random-effects model was favored over a fixed-effects model for our meta-analysis.We evaluated statistical heterogeneity with Cochran's Q test and quantified its extent using the I 2 statistic.Depending on the I 2 values, heterogeneity was classified as either insignificant (0%-25%), low (25%-50%), moderate (50%-75%), or high (75%-100%) (19).For transparency and procedural clarity, we registered our study protocol with the International Platform of Registered Systematic Review and Meta-Analysis Protocols (INPLASY) network (registration number INPLASY202380091).

Baseline patient characteristics
The analysis included 37 cohorts that collectively comprised 6475 diagnosed AML patients.Among these patients, 907 out of 5206 (17.42%) were identified as having AML with MS, resulting in 2199 MS cases.Table 1 details the characteristics and quality assessments of the analyzed cohorts.There was a slight male predominance, accounting for approximately 58.26%, with females comprising 41.74%.When analyzing age demographics across cohorts, 71.87% of the patient population was over 18 years old, while the remaining 28.13% was under 18.Further analysis indicated that the most common sites of MS manifestation were the skin, orbit, central nervous system, and lymph nodes.

Discussion
MS, commonly known as extramedullary AML, presents a wide range of clinical manifestations and often poses therapeutic challenges.A prior multicenter cohort study documented an MS incidence of 14.21% among newly diagnosed AML cases.Typically, pivotal therapeutic choices are guided by the genetic alteration profile.This current study marks the inaugural meta-analysis of MS prevalence and its associated genetic abnormalities.
Our study found a cumulative MS incidence of 17.42%, with a slight male predominance.This rate exceeds that reported in earlier research on newly diagnosed cases (10).Our finding aligns with the observations made by Fianchi et al. (51), who reported a decline in MS incidence from 11% to 7% when assessed at the time of AML diagnosis.In terms of molecular genetics, our meta-analysis identified FLT3-ITD mutations as the most frequently linked to MS, with a pooled prevalence of 17.5%.These figures align closely with prior research: Ansari-Lari et al.     patients.Another key finding from our study is that DNMT3A and TET2 mutations emerged as the third and fourth most common genetic aberrations, respectively.
AML with t(8;21)(q22;q22.1);RUNX1::RUNX1T1 represents a distinct subtype of AML.Classified as a core-binding factor leukemia, this form of AML is characterized by frequent genetic recurrence and generally has a favorable prognosis (54).Saia et al. delved into the RUNX1::RUNX1T1 rearrangement in mouse models, shedding light on its frequent association with extramedullary disease (55).Consistent with our data, the predominant fusion gene detected in MS was RUNX1::RUNX1T1, demonstrating a cumulative prevalence of 28.10%.This finding accords with the work of Hu et al. and Velagala et al., and it emphasizes the significance of the RUNX1:: RUNX1T1 fusion gene (5, 7).In contrast, certain studies have underscored the sporadic nature of the RUNX1::RUNX1T1 fusion, noting its presence in just 2% to 3% of MS cases (56).It is noteworthy that significant statistical heterogeneity was observed throughout all fusion gene analyses, likely amplified by varying baseline characteristics among the considered studies.
Geographical variations also influence the genetic profiling in MS.The NPM1 mutation was prominently observed among patients in Western regions, whereas KRAS predominated in those from Eastern countries.Additionally, FLT3-ITD was identified as a commonly occurring mutation in both populations.Core-binding fusion genes were frequently observed in AML with MS across continents, albeit with differences in specific fusion genes (CBFB::MYH11 in the Western population and RUNX1::RUNX1T1 in the Eastern population).
When comparing genetic abnormalities between non-MS and MS cases, our analysis revealed no significant differences in the prevalence of the RUNX1::RUNX1T1 and CBFB::MYH11 fusion genes.However, we found that the NRAS mutation was significantly associated with the MS group.Our findings suggest that the CEBPA mutation might confer a protective effect against MS, supported by an OR of 0.51 (95% CI 0.32 to 0.81).
This study examined the prevalence of mutations and fusion genes in AML with multiple MS.The findings revealed variations in the incidence rates of certain mutations between the MS group and AML patients without MS.Furthermore, age and geographical disparities emerged as significant factors influencing the genetic profiling in MS cases.Consequently, a mutational workup should be conducted in all newly diagnosed AML patients with MS, as the results offer valuable insights for risk stratification, guiding treatment decisions, and potentially introducing novel therapeutic options targeting specific mutations.
However, this study has several limitations.First, it drew upon published data, potentially introducing publication bias since studies with positive or novel outcomes are more likely to be published than those with negative or neutral findings.Second, numerous analyses indicated elevated I 2 values, which signify substantial heterogeneity among studies.This heterogeneity might have undermined the reliability of our combined results.Third, some included studies lacked details of patients' baseline characteristics, and there was evident statistical inconsistency in the genetic testing methods used.Additionally, the source of genetic data, whether derived from bone marrow or blood samples, was not always clearly specified, potentially leading to inaccurate representation of genetic variations.Fourth, the limited number of studies comparing gene mutations between MS and non-MS may have resulted in insufficient statistical power to establish significant differences.Fifth, the included studies utilized a range of techniques, such as conventional cytogenetics, Fluorescence In Situ Hybridization, polymerase chain reaction, and NGS, each with varying sensitivities in detecting mutations.This diversity in methods may have influenced the accurate assessment of mutation prevalence.Lastly, the potential relationship between MS and gene mutations is an intriguing area of study; however, we were unable to perform such an analysis in this study due to insufficient data.

Conclusion
This study underscores the importance of three gene mutations -FLT3-ITD, MLL, and NPM1-which were commonly observed in cases of MS.The fusion gene RUNX1::RUNX1T1 emerged as the principal genetic fusion associated with MS.Intriguingly, although the CEBPA mutation appeared to confer some protection against MS, the presence of the NRAS mutation was associated with an elevated risk of developing MS.In essence, this meta-analysis substantially augments our comprehension of the genetic mutation characteristics of MS.

FIGURE 1
FIGURE 1 (21) found these mutations in 15% of MS cases.Pemmaraju et al. (16) and Shallis et al. (3) also reported the FLT3 mutation as the predominant mutation.The second most common mutations were MLL and NPM1, with a pooled prevalence of approximately 17%, as highlighted by the studies of Chang et al. (52) and Eckardt et al. (10).According to Chang et al. (1), the MLL gene mutation, especially the classic 11q23 abnormality but excluding t(9;11), has been associated with extramedullary involvement and remains a poor prognostic factor.Additionally, Falini et al. (3) recorded NPM1 mutations in 14% of 181 MS samples.In a larger cohort of 89 AML patients, Ovcharenko et al. (53) observed mutated NPM1 in 13 out of 15 MS

5
FIGURE 5Forest plots showing the prevalence of fusion genes in AML patients with myeloid sarcoma in comparison to those without myeloid sarcoma.(A) RUNX1::RUNX1T1; and (B) CBFB::MYH11.

TABLE 1
Summary of baseline patient characteristics of each included article.

TABLE 2
Pooled prevalence of gene mutations in AML patients with myeloid sarcoma.