Sex-associated differences in frequencies and prognostic impact of recurrent genetic alterations in adult acute myeloid leukemia (Alliance, AMLCG)

Clinical outcome of patients with acute myeloid leukemia (AML) is associated with demographic and genetic features. Although the associations of acquired genetic alterations with patients’ sex have been recently analyzed, their impact on outcome of female and male patients has not yet been comprehensively assessed. We performed mutational profiling, cytogenetic and outcome analyses in 1726 adults with AML (749 female and 977 male) treated on frontline Alliance for Clinical Trials in Oncology protocols. A validation cohort comprised 465 women and 489 men treated on frontline protocols of the German AML Cooperative Group. Compared with men, women more often had normal karyotype, FLT3-ITD, DNMT3A, NPM1 and WT1 mutations and less often complex karyotype, ASXL1, SRSF2, U2AF1, RUNX1, or KIT mutations. More women were in the 2022 European LeukemiaNet intermediate-risk group and more men in adverse-risk group. We found sex differences in co-occurring mutation patterns and prognostic impact of select genetic alterations. The mutation-associated splicing events and gene-expression profiles also differed between sexes. In patients aged <60 years, SF3B1 mutations were male-specific adverse outcome prognosticators. We conclude that sex differences in AML-associated genetic alterations and mutation-specific differential splicing events highlight the importance of patients’ sex in analyses of AML biology and prognostication.


Patients and treatment
We analyzed 1726 adults with de novo AML, including 749 self-reported women and 977 men, who were treated on frontline Cancer and Leukemia Group B (CALGB)/Alliance for Clinical Trials in Oncology (Alliance) protocols.We limited our study to patients with de novo AML in order to avoid the confounding effects of AML type, that is, de novo AML versus AML evolving from an antecedent myelodysplastic syndrome versus therapy-related AML.Self-reported patient sex was confirmed by the results of centrally reviewed [25] metaphase karyotyping.Almost all patients received intensive cytarabine/anthracycline-based frontline treatment on CALGB/Alliance trials between 1986 and 2015 (details are provided in the Supplementary Information).Per study protocols, no patient received an allogeneic hematopoietic stem-cell transplantation (HSCT) in first complete remission (CR), and patients who received an offprotocol HSCT were excluded from analyses of disease-free (DFS) and overall survival (OS) because their follow-up data were either missing or incomplete.Because of differences in the treatment protocols between patients aged <60 years and those aged 60 years and older (Supplementary Information), we performed outcome analyses separately for these age groups.Institutional Review Board approval of all CALGB/Alliance and German AML Cooperative Group (AMLCG) protocols was obtained before any research was performed.Patients provided study-specific written informed consent to participate in treatment studies.Treatment protocols were in accordance with the Declaration of Helsinki.The AMLCG validation cohort of 954 patients (465 female and 489 male) is described in the Supplementary Information.

Mutational profiling
The mutational status of 80 protein-coding genes was determined centrally at The Ohio State University by targeted amplicon sequencing using the MiSeq platform (Illumina, San Diego, CA) [26].Testing for FLT3 internal tandem duplications (FLT3-ITD) was performed with the Sanger sequencing method [27].Determination of CEBPA bZIP status was done following 2022 European LeukemiaNet (ELN) guidelines [1] using Miseq panel and/or transcriptional profiling [28].SF3B1 and SRSF2 were considered as mutated if they concurred with the biology-associated differential splicing events (see details below).Experimental details are provided in the Supplementary Information.Preparation of samples and sequencing in the AMLCG validation cohort followed a comparable workflow [18].Methods used to analyze gene expression and differential splicing are described in the Supplementary Information.

Clinical endpoints and statistical analysis
Definitions of clinical endpoints-CR, early death, DFS and OS-are provided in the Supplementary Information [29].Pretreatment features of female and male patients were compared using the Fisher's exact for categorical variables and Wilcoxon rank-sum tests for continuous variables.Estimated probabilities of DFS and OS were calculated using the Kaplan-Meier method, and the log-rank test evaluated differences between survival distributions [30].We used logistic regression for modeling CR and Cox proportional hazard regression for interaction and modeling DFS and OS for univariable and multivariable outcome analyses, which were calculated using a limited backward selection technique, and adjusted P values to control for per family error rate.All analyses were performed by the Alliance Statistics and Data Center on a database locked on June 6, 2021, using SAS 9.4, TIBCO Spotfire S+ 8.2 and GraphPad Prism 9.
To validate the observed sex-associated differences, we compared the frequencies of cytogenetic findings (Supplementary Table 1) and gene mutations in patients from AMLCG (Tables 1 and  2).The results were largely concordant, with CN-AML and all mutations, except KIT and WT1 mutations, which differed between males and females among CALGB/Alliance patients being also significantly different in the AMLCG cohort.However, in the latter patient population, females also less often carried EZH2 (P = 0.005), SMC1A (P = 0.003) and STAG2 (P < 0.001) mutations and mutations in the cohesion complex genes (P < 0.001) than males.
The proportions of patients assigned to genetic-risk groups in the 2022 ELN classification [1] also differed between sexes.In the CALGB/Alliance cohort, a higher percentage of female patients was categorized in the intermediate-risk group (30% vs 20%) and a lower percentage of women was included in the adverse-risk group (31% vs 44%, Supplementary Table 1).

Sex-associated differences in co-occurring molecular alterations
We next compared patterns of co-occurring recurrent mutations in male and female patients and differences between sexes (Pearson correlation <0.2 and P < 0.0002 for one sex and Pearson correlation >0.03 and P > 0.5).We identified four gene mutation pairs, including NRAS/BCOR, U2AF1/CBL, NOTCH1/BCORL1, and SF1/ SRSF2 whose presence was strongly associated with female patients.Conversely, ASXL1/TET2, NF1/SF3B1, RUNX1/PHF6, RUNX1/U2AF1 and RUNX1/STAG2 were strongly positively associated with male patients.Conversely, WT1/SRSF2 tended to be mutually exclusive in males but less so in females (Fig. 1).

Treatment outcome of adults with AML aged <60 years with respect to patient sex
We analyzed clinical outcomes of 381 female and 463 male AML patients aged 17-59 years in the CALGB/Alliance cohort, for whom frontline treatment with intensive induction chemotherapy is currently still a standard of care.We found no significant differences in CR and early death rates, DFS or OS between female and male AML patients (Supplementary Table 2).There were also no differences between sexes in CR and early death rates or DFS in the AMLCG cohort.However, male German patients aged 18-59 years had a shorter OS than female patients (5-year rates, 42% vs 51%, P = 0.005).
Sex-associated prognostic impact of genetic alterations in patients aged < 60 years Since cytogenetic and molecular genetic findings are routinely used for risk stratification of AML patients, we assessed sexspecific outcomes based on the 2022 ELN genetic-risk categories [1].Despite differences in proportions of females and males in the intermediate-and adverse-risk groups, DFS and OS was essentially equal within each genetic-risk group, except for longer OS of female adverse-risk patients (P < 0.001; Supplementary Fig. 1A, B).Next, we assessed associations of recurrent genetic alterations with outcome of CALGB/Alliance patients.In univariable analysis (UVA) for CR, presence of FLT3-ITD, PTPN11 and RUNX1 mutations affected outcome of only female patients (Fig. 2A), of which FLT3-ITD and PTPN11 mutations remained significantly associated with CR achievement also in subsequent multivariable analysis (MVA, Supplementary Table 3).In contrast, a normal karyotype and SF3B1 mutations were adverse predictors only in male patients in both UVA and MVA.
For DFS in female patients, DNMT3A and WT1 mutations associated with shorter, and FLT3-TKD mutations with longer DFS in both UVA and MVA (Fig. 2B, Supplementary Fig. 2A).While for male patients detection of ASXL1, CEBPA bZIP , NRAS, SRSF2 and TP53 mutations had sex-specific survival impact in UVA, only CEBPA bZIP mutations were significant in multivariable models.
In univariable analysis of OS, FLT3-TKD, DNMT3A and WT1 mutations affected outcome of only female patients (Fig. 2C) of which, again, WT1 mutations held their significance also in multivariable analyses (Fig. 3A).Notably, the adverse outcome impact of female WT1-mutated patients seemed to be driven by WT1/NPM1 co-mutated female patients (Fig. 3B, C).OS of men only was influenced by CEBPA bZIP , GATA2, KIT, NRAS, SF3B1, SRSF2, STAG2 and U2AF1 mutations in UVA, of which SF3B1 mutations had again sex-specific survival association in MVA, too (Fig. 3D, Supplementary Table 3).Focusing on those sex-specific molecular alterations that held their significance in both UV and MV models and at least 2 survival endpoints (female: WT1 mutations [DFS, OS]; male: SF3B1 mutations [CR, OS]), we tested their outcome impacts in the AMLCG cohort.
While WT1 mutations associated with a trend for inferior DFS in female AMLCG patients, there was no association with inferior OS, possibly suggestive of a rescue effect of more frequently used intensive consolidation including allogeneic HSCT which is more commonly administered in Germany (Supplementary Fig. 2B, C).Although limited by relatively small sample sizes, SF3B1-mutated male patients in the AMLCG cohort also had a lower CR rate (38% vs 72%, P = 0.04) and tended to have inferior OS (Supplementary Fig. 3).
As SF3B1 mutations demonstrated distinct prognostic differences and were recently added to the 2022 ELN classification as adverserisk outcome prognosticator (in the absence of favorable-risk genetic markers), we compared OS of adverse-risk female and male patients with SF3B1 mutations and OS of the remaining adverse-risk female and male patients (i.e., without SF3B1 mutations).While male SF3B1mutated 2022 ELN adverse-risk patients and both female and male adverse-risk patients without SF3B1 mutations had similarly poor overall survival, female SF3B1-mutated adverse-risk patients had significantly longer OS (5-year rates, 43% vs 8%, P = 0.01) than other 2022 ELN adverse-risk patients (Fig. 3E).
Treatment outcome of adults with AML aged ≥60 years with respect to patient sex and the sex-associated prognostic impact of genetic alterations Given the generally poor survival of older patients with AML, we performed a subset outcome analyses of the AML patients aged 60 years and older included in our study.We did not find significant differences between sexes among 524 older patients treated on the CALGB/Alliance protocols nor among 414 older German patients (Supplementary Table 4).There were no significant differences in DFS or OS between female and male patients in any of the 2022 ELN The category "myelodysplasia-related" mutations has been included in the Table because it is used as an adverse-risk criterion in the widely adopted 2022 European LeukemiaNet genetic-risk classification [1,44].This category contains mutations in the SRSF2, SF3B1, RUNX1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR and STAG2 genes.genetic-risk groups, with patients in the intermediate-risk and adverse-risk groups performing similarly poorly (Supplementary Fig. 4A, B).Although drawing definitive conclusions from the multivariable analyses is difficult because of the generally poor treatment outcome of older patients with AML, we found that such mutations included in the 2022 ELN genetic-risk classification as FLT3-ITD, NPM1 and TP53 mutations were among main factors affecting CR rates and survival (Supplementary Table 5).Only in men, DFS was also negatively affected by PTPN11 mutations and OS by KRAS mutations.3 Overall survival of female and male patients with AML according to their WT1, NPM1 and SF3B1 mutation status.A Overall survival of female and male patients with and without WT1 mutations and overall survival of B female and C male patients according to their NPM1 and WT1 mutation status.D Overall survival of female and male patients with and without SF3B1 mutations.E Overall survival of female and male patients classified in the 2022 ELN adverse-risk group because of the presence of SF3B1 mutation (and lack of favorable-risk genetic markers) and of the remaining female and male patients classified in the 2022 ELN adverse-risk group who did not have SF3B1 mutation.

Sex-specific alternative splicing events and lineage-associated gene expression patterns
To investigate the potential role of sex bias on gene and splicing programs, we next analyzed patients with existing RNAseq data (n = 848) to quantify alternative splicing events (ASE) and differentially expressed genes (DEGs) associated with recurrent gene mutations or cytogenetic findings using the AltAnalyze workflow, including those affecting spliceosome genes and other recurrent AML-associated genes or cytogenetic features (n = 24) and select clinical/patient demographic variables.
Next, we compared female and male patients in each of these defined subtypes.We considered subtypes with >17 samples and an unadjusted P-value for these comparisons.Interestingly, we found different magnitudes of splicing and gene-expression impacts associated with sex in AML subtypes, with generally greater impacts associated with ASEs versus DEGs (Fig. 4C, D).In general, splicingfactor mutations (SRSF2-P95*, U2AF1-S34*, SF3B1, SF3A1, SF3A1, SF1) resulted in large sex-associated differences for both ASEs and DEGs.Notably, while it has been extensively documented that SF3B1-K700E and functionally similar mutations result in alternative splice sites only selected for within the presence of these mutations, examination of SF3B1-K700E and related mutation ASEs predicted sex bias for several of previously annotated (USP25, MYO15B) and novel SF3B1-K700E selected cryptic splice sites (AL133352.1,CNTRL, DOCK10), in addition to non-cryptic splice sites with clear sex bias (Fig. 4E).At the pathway level, we found that SF3B1 mutations in male patients transcriptionally induce genes associated with integrin cell-surface receptor-linked signaling pathways (MAP kinase, Netrin, PECAM1, Ephrin B, interferon alpha/beta signaling, L1CAM), whereas RUNX1and TP53-mutated male patients induce inflammatory response pathways (IL12, FGF, CXCR4 and TCR signaling).Further, complex karyotype and TP53 mutations in female patients induce a distinct set of inflammatory and immune signaling programs (Supplementary Fig. 5C, D).When we considered marker genes for hematopoietic human progenitor and differentiated cell populations, we noted distinct lineage enrichments shared by different sets of mutations.In particular, SF3B1 (females) and KRAS (males) were enriched in hematopoietic stem cells, MultiLin, common lymphoid precursor and eosinophil/mast programs, whereas SF3B1 (males) and WT1 (females) induced a dominant megakaryocytic progenitors/erythroid progenitors (Fig. 4F).We note that in male SF3B1-mutated patients, genes associated with these programs represent well-defined lineage determining factors, such as HOPX and PROM1 for hematopoietic stem cells, ITGA2B, VWF and PF4 for megakaryocytic progenitors and EPOR and GATA1 for erythroid progenitors (Fig. 4G).Together, these data indicate that sex contributes to both alternative splicing and transcriptional differences among patients with splicing factor and other dominant AML mutations.
Recently, De-Morgan et al. [23] also demonstrated specific preleukemic genes that were more frequently mutated in men than women with AML.Consistent with previous reports, our study found a female predominance of such most common (de novo) AML-associated mutations as NPM1, DNMT3A and FLT3-ITD, and a male predominance of spliceosome complex gene mutations and other myelodysplasia-related genes, many of which are found on the X-chromosome (BCOR, BCORL1, SMC1A, STAG2, ZRSR2).These findings might be interesting in the context of the known higher incidence of AML in males compared to females, and suggestive of sex-specific differences in the disease biology.Interestingly, analyzing gene expression and alternative splicing patterns first on a global scale followed by sex-specific analyses, we identified differential splicing events that associated with sex, as well as cell lineage, depending on the gene mutation.Surprisingly, our data suggest that shared pathway-level impacts for pairs of mutations exist, which tend to be associated with sex, whereas cell-type associated impacts indicate a lineage bias in programs, which often differ from the pathway.Although biologic reasons for the male-specific outcome association of SF3B1 mutations are still unclear, the differential effects on cell lineages suggested by GSEA indicate that sex-specific cell programs influencing leukemogenesis and leukemic cell responses may exist.
We detected male-specific association of SF3B1 mutations with higher resistance to induction chemotherapy and shorter overall survival.Notably, SF3B1 mutations, in the absence of favorable-risk genetic markers, have recently been added as an adverse-risk prognosticator to the 2022 ELN genetic-risk classification [1].Our data expand upon the work of the GenoMed4All consortium and Maggioni et al. [33], which also demonstrated sex-specific biases at the single-gene level in myelodysplastic syndromes (MDS) and a male specificity for co-mutational pathways in splicing-related genes.Our work, if confirmed, ultimately suggest that SF3B1 mutations might constitute a sex-specific prognosticator, which could be taken into account in the future revisions of the ELN genetic-risk classification.
The negative prognostic impact of WT1 mutations has been repeatedly reported in the past [34][35][36][37][38][39], but their association with female sex have hitherto not been recognized.In our study, an association of WT1 mutations with women was seen only in the CALGB/Alliance cohort, but was not observed in the AMLCG patients.As patients treated in Europe receive allogeneic HSCT in first CR more often, it is possible that more intensive consolidation might alleviate the adverse prognostic impact of WT1 mutations.This discrepancy might warrant further investigation in prospective studies, to help determine the appropriate consolidation strategy for female patients carrying WT1 mutations.Moreover, adverse outcome of WT1-mutated women seems to be associated with co-existence of NPM1 mutations, which was previously suggested to confer high treatment resistance and poor survival rates [17,40].
Limitations of our study include the fact that we studied only patients with de novo AML, and that the Alliance patients did not undergo allogeneic HSCT in first CR and were almost exclusively treated with intensive induction chemotherapy followed by consolidation therapy.Hence, future studies are needed to assess associations between sex and molecular features and survival in patients undergoing allogeneic HSCT and in those receiving emerging targeted therapies, which is particularly important in older patients.Furthermore, we detected sex-associated differences in the frequencies of myelodysplasia-related gene mutations among patients diagnosed with de novo AML, not in patients with AML evolving from an antecedent MDS whom we did not analyze.It will be thus important to perform dedicated analyses in patients with secondary AML evolving from MDS, especially since sex disparities with respect to genotypes, phenotypes and outcomes have previously been reported in patients diagnosed with MDS [41,42].
In summary, our study assessed potential sex-associated biologic and prognostic implications of genetic alterations in large patient cohorts.Although most established genetic alterations affected outcomes of both sexes, select gene mutations were associated with patient sex.This was likely missed previously because of routinely performed sex-pooled outcome analyses.We identified SF3B1 mutations as a male-specific and WT1 mutations as female-specific prognosticators of poor survival in patients treated with conventional chemotherapy.Differences in the frequencies of AML-associated genetic alterations and mutationspecific differential splicing events, with possible subsequent phenotypic changes, highlight the importance of considering the patients' sex in analyses examining leukemia biology and prognostic significance of genetic alterations and their role in clinical decision-making in AML.
Fig. 4 Alternative splicing events (ASEs) segregated by mechanism of predicted regulation for recurrent mutations, gene fusions and clinical covariates in the CALGB/Alliance RNA-Seq cohort.The extent of exon/intron A inclusion or B exclusion ASEs.The extent of unique sex-associated ASEs C or differentially expressed genes (DEGs) D associated with each patient subtype, unique regulated events or genes compared with all other AMLs and sex/subtype overlapping events/genes, derived from the software AltAnalyze.Green color denotes the number of unique ASEs or DEGs in all patients with the indicated molecular genetic or cytogenetic subtype versus all other AML patients in the cohort.Blue indicates the number of unique ASEs or DEGs in males versus females in the indicated AML subtype and red illustrates overlap between subtype-associated ASEs or DEGs and male versus female (subtype regulated events/genes that vary according to patient sex).E Quantification of SF3B1-mutated sex-associated alternative splicing events as percent spliced in (PSI) values for the indicated parental exon-exon junction.F = female, M = Male.F Heatmap of gene set enrichments (GO-Elite) for known AML subtypes against human bone marrow cell-type markers to identify lineage skewing, BM cell-type markers were derived from extensive prior human single-cell analyses [43], to identify markers of rare hemopoietic stem cells, progenitors, and immune cell-types.Only AML subtype sex-associated gene sets with significant enrichments are shown.G Sex-associated differentially expressed genes in SF3B1-mutated males versus females corresponding to human bone marrow progenitor populations.

Fig. 1
Fig.1Differences in co-existing molecular features between female and male patients with AML.Triangle correlation plot depicting cooccurring mutations in A female and B male patients with AML treated on CALGB/Alliance frontline protocols.All squares denoting gene mutation pairs that differ between the sexes are marked using stars.

Female 35 NPM1Female
Fig.3Overall survival of female and male patients with AML according to their WT1, NPM1 and SF3B1 mutation status.A Overall survival of female and male patients with and without WT1 mutations and overall survival of B female and C male patients according to their NPM1 and WT1 mutation status.D Overall survival of female and male patients with and without SF3B1 mutations.E Overall survival of female and male patients classified in the 2022 ELN adverse-risk group because of the presence of SF3B1 mutation (and lack of favorable-risk genetic markers) and of the remaining female and male patients classified in the 2022 ELN adverse-risk group who did not have SF3B1 mutation.

Table 1 .
Frequencies of mutations in single genes in female and male patients with AML treated on the CALGB/Alliance frontline protocols and those treated on the German AML Cooperative Group frontline protocols.

Table 1 .
continued *P values for categorical variables are from Fisher's exact test, P values for continuous variables are from the Wilcoxon rank-sum test.

Table 2 .
Frequencies of gene mutations arranged in functional groups in female and male patients with AML treated on the CALGB/Alliance frontline protocols and those treated on the German AML Cooperative Group frontline protocols.
* P values for categorical variables are from Fisher's exact test, P values for continuous variables are from the Wilcoxon rank-sum test.a Chromatin remodeling is mutated if one of ASXL1, BCOR, or EZH2 is mutated.Kinases is mutated if one of FLT3-ITD, FLT3-TKD, or KIT is mutated.Methylation is mutated if one of DNMT3A, IDH1/2, or TET2 is mutated.RAS pathway is mutated if one of CBL, KRAS, or NRAS is mutated.Spliceosome is mutated if one of SF3B1, SRSF2, U2AF1 or ZRSR2 is mutated.Transcription is mutated if one of CEBPA, ETV6, IKZF1, GATA2 or RUNX1 is mutated.Tumor suppressor is mutated if PHF6, TP53 or WT1 is mutated.b