Bravo‐Pérez, Carlos

Abstract Myeloid neoplasia encompasses a heterogeneous and complex molecular landscape whereby the distinct combinatorial patterns associated with clinical phenotypes are difficult to delineate. Given that genetic heterogeneity can be a consequence of the topology of the fitness landscape (peaked/non-peaked=high vs low fitness), we investigated co-mutational patterns in TP53 context as an example of hallmark genomic heterogeneity. Indeed, TP53 gene is characterized by hypomorphic/loss-of-function lesions, truncating mutations (frameshifts, stop codons), missense mutations often having a dominant-negative effect and loss of heterozygosity by chromosome 17p deletion driving clonal expansion. Clinical phenotypes of TP53 mutants are also heterogeneous. Here, we stratify TP53 mutations based on our previous published data1 whereby patients with single-hit TP53 and variant allelic frequency (VAF) >50% were classified as obligatory biallelic, patients with VAF between 23-50% as probable biallelic and <23% as probable monoallelic. A threshold of VAF 23% was optimal to separate monoallelic vs biallelic and was based on all-cause mortality. A tree-based model (RandomForestSRC) was used to build a survival model with VAF as a single covariate and minimum node size set to 15 to optimize the cut-off. Using this cut-off, we delineate the epistatic landscape and potential interactions among different TP53 mutational spectra and its fitness. We curated a cohort of 800 patients with TP53 mutations from our institution. In total 36% of the cases carried single hits while 64% had double hits genomic configuration. About 70% of the mutations were missense and were detected at canonical sites (R175H, Y220C, M237I, R273H, R282W, R248Q). We took into consideration 35 features and clustered our cohort of TP53 mutants using factor analysis. When we stratified the cohort based on our predetermined VAF threshold, we found that majority of the variants with high VAF, to be deemed as obligatory biallelic, co-occurred with complex karyotype. These cases were very homogeneous hence had on average a low number of additional variants, likely due to TP53 clones having reduced fitness in the presence of additional mutations. Conversely, patients without complex karyotype were mostly assigned to the monoallelic group due to low to median VAFs. Consequently, these samples had on average a higher number of additional hits, hence a heterogeneous molecular landscape. To substantiate this finding, we noticed that among monoallelic patients two characteristic pairs were observed (TP53-TET2 and TP53-SF3B1) and showed different patterns according to karyotypic associations. Indeed, both pairs in normal karyotype (TP53-TET2, 65%; TP53-SF3B1, 23%) had the highest number of additional mutations on average compared to other monoallelic hits in complex karyotype suggesting that clones resulting by these pairs were advantageous and produced higher fitness driving heterogeneity when together with other mutations. As a preliminary analysis to characterize the fitness landscape of canonical missense TP53 mutations, we investigated whether distinct TP53 mutations conferred differential clonal fitness by implementing a previously published model of stochastic hematopoietic stem cell growth based on the distribution of allelic frequencies across different age groups. Given a fixed stem cell population pool and a constant mutation rate, the distribution of allelic frequencies in the population of corresponding mutations across age can inform the fitness/selection coefficient. The model predicts that high fit mutations will expand more in the population (high VAF) depending on the time the mutation is allowed to expand (age). Based on the literature, we set the stem cell pool to 100,000 and the mutation rate to 1.4E-8.2We limited our analysis to mutations found in at least 10 individual in our cohort and to single hits due to potential epistatic interactions across multiple mutations in the same gene. Given this criterion two mutations R273H and R248Q were analyzed. Interestingly, we observed a 15% increase in fitness gain for R273H compared to R248Q. In summary, mutational heterogeneity and clonal fitness might provide clues to the diversity of TP53 mutational configurations. Our model will be further optimized for the upcoming ASH meeting by incorporating temporal sampling and increasing sample size to define differences in fitness according to mutation site.

Abstract Chronic idiopathic neutropenia (CIN) is characterized by a persistently reduced absolute neutrophil count, typically <1.5×10⁹/L for ≥3 months, without identifiable infectious, nutritional, autoimmune, clonal, congenital, or drug-related causes. It most commonly affects adult women, carries a variable risk of infection, and is frequently managed expectantly in the absence of severe or recurrent infections. An index case of CIN with irregular menses and normalization of counts during pregnancy prompted an institution-wide review of adult neutropenia. U[p front we excluded cases of known congenital and drug-induced neutropenia. Among 238 presumed CIN patients, 88 had large granular lymphocytic (LGL) leukemia–associated neutropenia, 63 had autoimmune neutropenia defined by the presence of anti-neutrophil antibodies, and 87 met criteria for idiopathic neutropenia. Within the presumptive CIN group, extended blood antigen screening identified 12 patients with a Duffy-null phenotype, who were excluded from further idiopathic analysis (final n=75). This included 10 African American, 1 Caucasian, and 1 Asian patient. Extended blood-group profiling (Kidd, Colton, Lutheran, Dombrock, LW, ABO) showed no significant deviation from population frequencies. In the remaining non-Duffy CIN patients, remarkably 54/75 were female with a mean age of 45±16 years. At presentation, mean WBC was 2.84±0.58×10⁹/L and ANC was 0.92±0.39×10⁹/L, Hgb was 13.5±1.2g/dL and platelets were 229±83×10⁹/L. Antinuclear antibodies were assessed in 73 patients, with 63 negative and 10 positive, predominantly speckled (66%) but rheumatologic consultation did not yield systemic autoimmune diagnoses. Hypothyroidism was noted in two CIN cases. Irregular menses were reported in 50% of female CIN patients with documented histories, and among those, 80% exhibited higher or normal neutrophil counts during gestation. Physiologically, pregnancy is a progesterone-high state associated with neutrophilia and physiologic leukocytosis; in addition, progesterone can dampen cytotoxic T-cell activity and inflammatory signaling while supporting marrow niche function. Furthermore, progesterone binds PR on marrow stromal cells and can upregulate CSF3 (G-CSF) and possibly other hematopoietic growth factors, thus regulating granulopoiesis. By contrast, anovulatory or irregular cycles often reflect luteal progesterone deficiency, which may predispose susceptible patients to lower ANC prompting us to formulate a theory that excess of CIN in females may be due to a progesterone-responsive CIN subtype. From our cohort, there were no patients on progesterone-supplement to evaluate if there would be responsiveness secondary to its utilization. To further support this theory we turned to metanalytic resources. TriNetX1allowed us search and identify women receiving exogenous progesterone versus a control receiving non-hormonal contraceptives and assign to them a corresponding ANC. When we compared ANC in 1:1 greedy propensity-score matching to control for covariates (demographics, BMI, and disease confounders e.g., malignancy). balance between comparator groups was confirmed by standardized mean differences <0.10. After matching (n=18,561 per arm), the exogenous progesterone group had a significantly higher neutrophil count compared to non-hormonal contraception (1.9±3.7 vs 1.5±1.7; t=3.5, p=.0004). The cohort age range was similar to our patient population 44 ±19. Our observations motivate prospective evaluation of progesterone-high states as potential modifiers of ANC in otherwise idiopathic cases and particularly refractory cases Cycle-synchronized CBC with paired progesterone and estradiol measurements, together with controlled progesterone augmentation trials, can quantify ANC changes and clinical outcomes in this subset. We thank TriNetX and all participants for their contribution, without whom this research would not have been possible. ReferencesRobertson SA, Mayrhofer G, Seamark RF. Ovarian steroid hormones regulate granulocyte-macrophage colony-stimulating factor synthesis by uterine epithelial cells in the mouse. Biol Reprod. 1996 Jan;54(1):183-96. doi: 10.1095/biolreprod54.1.183. PMID: 8838016.