Abstract We explored the impact of luspatercept therapy on overall survival (OS) and possible predictors of response in low‐risk (LR) myelodysplastic syndrome (MDS) patients. We evaluated 331 anemic patients treated with luspatercept. Hematological response (HI) was defined as (i) hemoglobin (Hb) increase of ≥1.5 g/dL in nontransfusion‐dependent (NTD) patients, and (ii) red blood cell (RBC) transfusion independence (TI) with a concomitant Hb increase of ≥1.5 g/dL, or RBC‐TI without an Hb increase of 1.5 g/dL, or >50% reduction in RBC transfusion burden (TB) for TD patients. Response was observed in 166 patients (50.2%), with significantly higher response in NTD and low TB versus high TB patients (p < 0.001). A significant correlation between lower Molecular International Prognostic Scoring System (IPSS‐M) risk scores and response was observed. No statistically significant difference in HI was found in SF3B1‐mutated versus wild‐type MDS patients (53.8% vs. 40.1%, respectively). SF3B1mut hotspots (K700E vs. others) and variant allele frequencies (VAFs; <38% VAF vs. ≥38% VAF) did not impact on HI. SF3B1‐mutated MDS with del5q showed inferior HI compared to other LR‐MDS (p = 0.046). The median treatment duration overall was 35 weeks (20.86–90.29), the median time to response was 11 weeks (8.71–21.86), and the median duration of response was 65 weeks (26.5–114). After a median follow‐up of 13 months, median OS was not reached (NR) for responders and 24 months for nonresponders (hazard ratio [HR] 0.25, 95% confidence interval 0.14–0.44, p < 0.001). This analysis of 331 luspatercept real‐life‐treated LR‐MDS patients demonstrated a significant OS benefit upon luspatercept response. Low baseline RBC‐TB and lower risk IPSS‐M scores correlated with higher HI and could constitute predictive markers of response.

ABSTRACT:Acquired somatic mutations are incorporated in the classification and prognosis of myelodysplastic syndromes/neoplasms (MDSs). However, the predictive role of molecular features in MDS needs to be elucidated, especially in the lower-risk subtypes (LR-MDS), where treatment has become heterogeneous and predictive biomarkers are lacking. In this study, we investigated genetic markers associated with erythropoiesis-stimulating agents (ESAs) response in LR-MDS. A European cohort of 535 patients with LR-MDS was analyzed using targeted next-generation sequencing (t-NGS) to calculate molecular prognostic scores (International Prognostic Scoring System, molecular [IPSS-M]). The integration of IPSS-M score among the 2 known variables, serum erythropoietin (sEPO) and transfusion dependence (TD), refined the capability to predict response (area under the curve [AUC], 0.71 vs 0.63, P = .0004). Based on these 3 variables, a molecular predictive score, which we named ESA-PSS-M (-0.05 × [sEPO U/L] -4.5 × [IPSS-M score] -5 × [TD (yes = 1; no = 0)]; specificity 76%; sensitivity 57%), was generated and validated in an external cohort (n = 223 patients with LR-MDS). Despite the impact of IPSS-M score, no single mutated gene was linked to ESA response; however, when we stratified cases by sex at birth, the X-linked STAG2 gene mutations were significantly associated with ESA resistance in males with LR-MDS (odds ratio, 0.13; P = .003). To our knowledge, this is the first study based on a large multicenter cohort of patients suggesting that the integration of IPSS-M score and sex-specific mutations can characterize ESA resistance and guide first-line (1L) therapeutic choices for anemic LR-MDS (ie, ESAs vs luspatercept).

INTRODUCTION Myelodysplastic syndromes with ring sideroblasts (MDS-RS) are typically associated with SF3B1 mutations (SF3B1MUT) and carry a favorable prognosis. However, approximately 20% of MDS-RS are SF3B1 wildtype (SF3B1WT), often harboring adverse genetics and poor outcomes even in the case of bone marrow (BM) low blast count (LB, <5%). In such cases, the presence of RS can be misleading. Therefore, this study aimed at identifying readily accessible clinical or morphologic markers predictive of SF3B1WTto detect these “high-risk” MDS-RS-LB cases, particularly in centers without access to molecular diagnostics. PATIENTS AND METHODS We analyzed MDS-RS cases from the IPSS-M public dataset, excluding those with del(5q) and with increased blast counts (learning cohort). RS positivity was defined according to WHO criteria (≥5% if SF3B1MUT, ≥15% without any secondary RS causes if SF3B1WT). We compared clinical, morphologic, cytogenetic, and mutational features between MDS-LB-RS SF3B1MUT vs SF3B1WT using univariate and multivariable logistic regression analysis. Overall survival (OS) and progression free survival (PFS) were also compared. In cases from the MDS Unit cohort (part of the validation cohort), flow cytometry was used to confront the myeloid, lymphoid and erythroid compartments between the two groups. Additionally, in MDS-LB-RS cases with IPSS-R ≤ 3.5, erythroid response to erythropoiesis stimulating agents (ESAs) and luspatercept (Luspa) was compared adopting IWG 2018 criteria. Finally, a predictive score for SF3B1 status was developed based on clinical and morphological parameters and validated in an external multicenter cohort composed by cases from MDS Unit (University of Florence), RESMD Spanish Registry and Argentina. RESULTS A total of 457 cases with MDS-LB-RS were identified from the IPSS-M dataset. SF3B1WT cases (n=87) were younger (mean 69.8 vs 72.3 y, p=0.03), more frequently had therapy-related MDS (11% vs 4.3%, p=0.01), and had significantly worse OS (median 3.8 vs 7.2 years, p<0.0001) and PFS (78% vs 91% at 3 years, p<0.0001) compared to SF3B1MUTcases (n=370). Although anemia severity was similar, SF3B1WT cases had lower platelet counts (149 vs 264 ×10⁹/L, p<0.0001), neutrophil counts (2.4 vs 3.2 ×10⁹/L, p<0.0001), fewer RS (36% vs 49%, p=0.01), and more frequently multilineage dysplasia (MLD, 76.5% vs 40%, p=0.0001). SF3B1WT cases were also enriched for poor-risk cytogenetics (8.9% vs 1.1%, p=0.008), complex karyotype (16.4% vs 0.3%, p=0.0001), and mutations in TP53 (28% vs 4.3%, p=0.001), U2AF1 (25.8% vs 1.3%, p=0.001), and SRSF2 (30.6% vs 1.8%, p=0.002), resulting in higher IPSS-M scores (0.14 vs –1.1, p<0.0001). Multi-hit (MH, defined as per WHO5th) TP53 status was more frequent (10% vs 0.5%, p=0.0001), and frameshift variants exclusive to SF3B1WT cases (29% vs 0%, p=0.002). Flow cytometry (n=37) showed that SF3B1WT cases (n=18) had fewer CD117⁺ erythroid precursors (30.6 vs 41.3%, p=0.03) and more BM monocytic cells (8.1 vs 3.9%, p<0.0001) than SF3B1MUTcases (n=29). Additionally, SF3B1WT cases showed a trend towards lower response rates to both ESAs (25% vs 55.6%, p=0.09) and Luspa (17% vs 39%, p=0.3). Multivariable analysis confirmed younger age (OR 0.973, p=0.038), lower platelet counts (OR 0.986, p<0.001), and presence of MLD (OR 0.354,p=0.002) as independent predictors of SF3B1WT. Based on these findings, we developed a score using age, platelet count and presence of MLD to predict SF3B1WT MDS-LB-RS forms: 0.15 x Age + 5 x MLD (1/0) + 0.05 x Plt (109/L). A cutoff < 23.05 identified SF3B1WT cases with 83.3% sensitivity and 75.2% specificity (AUC 0.83). This score was validated in the external cohort (SF3B1MUT n=238, SF3B1WT n=105), showing a similar good performance (sensitivity 77.2%, 80.3% specificity, AUC 0.82). CONCLUSIONS SF3B1WT MDS-LB-RS cases display distinct features, including enrichment of MH and frameshift TP53 mutations, and are associated with worse OS and PFS. In case of RS presence, if Perls staining is routinely performed, a simple score based on age, platelet count, and MLD can efficiently predict SF3B1WT forms, especially valuable in settings without molecular diagnostics. It would be interesting to evaluate the prognostic value of the score. Infact, these patients warrant closer monitoring due to their adverse genetic profile and inferior prognosis. Furthermore, their apparent lower response to standard anemia therapies suggests a need for alternative treatment approaches.

Immune dysregulation is increasingly recognized as both a pathogenic driver and therapeutic target in Myelodysplastic Syndromes/Neoplasms (MDS). Despite improvements in prognostic stratification by the Molecular International Prognostic Scoring System (IPSS-M), MDS patients remain heterogeneous in their clinical behaviour, treatment responses and disease evolution. Inflammation impact seems to be preponderant in pathophysiology of lower risk (LR)-MDS. By assessing 36 analytes in LR-MDS serum at diagnosis with a screening panel, we stratified and characterized LR-MDS cases into biologically distinct subgroups with different clinical characteristics and treatment response to erythropoietin stimulating agents (ESAs). We evaluated 95 MDS cases selected per lower risk IPSS-R (score < 3.5), and symptomatic anemia, analyzing 36 serum proteins (cytokines, alarmins, soluble receptors and growth factors). Complete clinical annotations (including IPSS-M scores) were collected. Serum protein levels were assessed using custom Luminex Screening Assay panels. For bioinformatic analysis, proteins were regrouped into simplified biologically relevant groups: pro-inflammatory (S100A8/A9, TNF-α, IL-1β, IL-18, IL-17E, IL-8, IFN-α/γ, CCL2/3/4, RANTES, CCL11, CXCL9/10/7/4; n=18), anti-inflammatory (IL-1RA, IL-10; n=2), bimodal (pro- and anti-inflammatory potential; IL-6, IL-4, IL-13; n=3), growth factors (VEGF, PDGF-BB, HGF, FGF-BASIC, M-CSF; n=5), receptors (TNFR1, CD25, GP130; n=3), apoptosis-related (TRAIL; n=1), and immune expansion (IL-2, IL-5, IL-7, IL-15; n=4). Following log-transformation and z-score normalization, we performed principal component analysis (PCA) and k-means clustering to identify distinct LR-MDS subgroups. Feature selection by random forest models identified the most discriminatory cytokines in LR-MDS. Subgroups were compared in terms of inflammatory state, clinical variables, and clinical endpoints including overall survival (OS) and ESA response duration. Median age at diagnosis was 74 years (range 47-91) (M:F ratio 1.6:1). Per WHO 2022 classification (n=69): MDS-LB = 44, MDS-SF3B1 = 21, and MDS-del(5q) = 4. IPSS-R scores (n=95) were: very low = 31, low = 55, intermediate = 9. IPSS-M scores (n=62): very low = 16, low = 32, moderate-low = 9, moderate-high = 4, and very high = 1. Through PCA we identified five distinct clusters based on serum protein levels. Cluster sizes were: C1 (n=18), C2 (n=17), C3 (n=12), C4 (n=22), and C5 (n=26). Inter-cluster comparisons revealed significant differences between all analytes annotated for biological function (adj. p < 0.001, except for receptors and apoptosis-related groups), especially for bimodal and pro-inflammatory proteins, and growth factors. Random forest analysis identified IL-4, IL-17E, IL-2, CCL3, IL-1β, IL-7, CCL4, IL-13, IFNγ and CXCL7 as most discriminatory serum proteins. Cluster-specific characteristics: C1 (72% low IPSS-R, median age 76 years) showed low pro-inflammatory levels (mean log10: 1.88); C2 had the highest MDS-del(5q) frequency (18%) and highest inflammatory burden (mean log10: 2.30, 3.2-fold higher than C3); C3 was the youngest (median age 58.6 vs 76.6 years overall, p=0.041), enriched for MDS with ring sideroblasts (MDS-RS, 33%), and displayed the lowest pro-inflammatory profile (mean log10: 1.80). There was a numerical difference in rate and duration of ESA response, with a trend to longer ESA response in C4 and C5 (log-rank p=0.089). Interestingly, clusters C1, C2 and C3 had higher rate of resistance or early relapse to ESA therapy (56%, 50%, and 67%, respectively) when compared to clusters C4 and C5 (18% and 23%, respectively). While overall survival did not significantly differ across clusters, cluster C2 showed higher rate of AML transformation (14% vs 6% non-C2). By profiling serum analytes in 95 LR-MDS patients we identified five biologically distinct LR-MDS subgroups with differential inflammatory signatures correlating with ESA responses. Extreme pro-inflammatory profile (C2), and low inflammatory profiles (C1 and C3) identify LR-MDS cases refractory to ESAs. In C2 poor response may be correlated with a more severe disease course, with AML early progression; while C3 is enriched for young MDS cases with low-inflammation burden with MDS-RS. These results are relevant to support early therapeutic decision-making and prompt further studies in larger LR-MDS cohorts to dissect cytokine-mediated molecular pathways.

AbstractRelevance of germline (GL) predisposition in myelodysplastic syndromes (MDSs) was stressed in both 2022 WHO and International Consensus classifications, but its incidence is probably underestimated, especially in young adult patients. We selected a cohort of 31 consecutive de novo MDS patients with unusual young age (<60 years). We performed exome sequencing (ES) on DNA extracted from noninvasive sources (peripheral blood and saliva), filtering for a panel of 344 genes specifically tailored for detecting GL variants related to clonal and nonclonal cytopenia. We observed at least one high‐ or low‐confidence GL MDS variant in 7/31 (22.6%) and 9/31 (29.0%) of cases, respectively. Four of 31 patients (12.9%) confirmed having established MDS/AML predisposing disorders. We found heterozygous variants in genes involved in DNA repair/cancer predisposition (ATM, ATR, FANCM, PARN, BRCA1, BRCA2, CHEK2, MSH2) in 9/31 (29.0%) cases and variants affecting ribosome biogenesis (SBDS), hematopoietic stem cell (GATA2), and megakaryocyte (ANKRD26) differentiation in single cases. Two cases had variants in RBBP6, a gene previously described exclusively in familial myeloproliferative neoplasms. Lastly, four cases had variants in genes related to inherited anemias (CUBN and PIEZO1 genes). Our results showed that “young” MDS patients aged 40–60 years carried reported and unreported GL variants with an unexpectedly high proportion, and these events co‐occurred with somatic mutations recurrent in myeloid neoplasms. We explored the “no man's land” of the young adult MDS cases adopting a practical and scalable diagnostic tool, capable to detect GL variants avoiding invasive methods.

Introduction Ageing is a major risk factor for the development of Myelodysplastic Syndromes/Neoplasms (MDS). Epigenetic alterations are common in MDS, especially aberrant DNA methylation. On a different level, DNA methylation at specific genomic loci can be used to capture the epigenetic age of cells or tissues. Epigenetic age acceleration (EAA) can thus be evaluated in individuals comparing biological age, obtained by specific tools called epigenetic clocks, with their chronological age. We assessed EAA in lower-risk MDS (LR-MDS) patients in parallel with serum cytokine levels and clinical/molecular variables, in the attempt to correlate biological ageing with inflammation and disease progression. Methods We evaluated 101 MDS cases diagnosed according to WHO 2022, selected per lower risk IPSS-R score. IPSS-M score was also calculated. Complete clinical annotations were collected. DNA was extracted from bone marrow mononuclear cells and assessed for DNA methylation using the Infinium MethylationEPIC v2.0 array. Data preprocessing was conducted in R using the Bioconductor package SeSAMe. Clock estimates of well-established epigenetic clocks (Horvath1, Hannum, DNAmPhenoAge, GrimAgeV1, and GrimAgeV2) and their principal-component counterparts were calculated using online tools. Unsupervised hierarchical clustering with Ward D2 algorithm was used to identify clusters of differential EAA. These clusters were compared across clinical variables, cytokine levels, and mutational landscape. Results Median age at diagnosis was 75 years (yrs) (36-91) (M:F ratio 2:4). Per WHO classification: 67 patients were MDS-with low blasts (LB); 13 MDS del(5q); 11 MDS SF3B1mut; 1 MDS-hypoplastic; 6 MDS-increased blasts-1; 1 MDS-increased blasts-2; 2 MDS-RS. IPSS-R scores were: very low = 38; low = 50; intermediate = 13. IPSS-M scores: very low = 24; low = 42; moderate low = 12; moderate high = 3; high = 5; not available = 15). Patients exhibited increased EAA in several epigenetic clocks: Horvath1 (mean 6.54 yrs), Hannum (2.71 yrs), GrimAgeV1 (2.39 yrs), and GrimAgeV2 (2.70 yrs), but not in DNAmPhenoAge (-0.60 yrs). Principal component clock counterparts also revealed increased EAA: PCHorvath1 (7.40 yrs), PCHannum (6.90 yrs), PCPhenoAge (7.65 yrs), and PCGrimAge (2.57 yrs). DNAmPhenoAge EAA positively correlated with IPSS-M (Spearman r=0.28, p = 0.013; Very low = -7.09 ± 13.55 yrs; low = -0.45 ± 11.68 yrs; moderate low = 4.16 ± 12.94 yrs). None of the epigenetic clocks correlated with IPSS-R scoring. Through unsupervised clustering analysis, we identified three distinct clusters based on EAA: Cluster A, that we defined as slow-agers, exhibited the lowest EAA across all evaluated clocks (n = 38; M:F ratio 1.7); cluster B displayed heterogeneous EAA (n = 42; M:F ratio 3.2); and Cluster C, termed fast-agers, showed the highest EAA across all clocks (n = 21; M:F ratio 2.5). Fast-agers show significantly lower serum CXCL10 (20.75 vs. 38.08 ng/mL) levels (p = 0.041, Wilcoxon rank sum) and a trend towards lower levels of senescence-associated secretory phenotype (SASP) factors compared to slow-agers, including CCL3 (51.54 vs. 69.48 ng/mL), CCL4 (108.00 vs. 219.75 ng/mL), IL6 (2.41 vs. 3.07 ng/mL), IL8 (12.92 vs. 34.89 ng/mL), TNF-α (4.08 vs. 6.32 ng/mL), and VEGF (51.94 vs. 62.89 ng/mL). By clinical variables (e.g., Hb, WBC, platelets, MCV) we could not define significant differences comparing slow - and fast-agers (cluster A and C). MDS SF3B1mut were predominantly fast-agers, while MDS with del(5q) and MDS-LB were more commonly slow-agers. There was no difference in progression to HR-MDS. With the limitation of few cases observed, fast-agers seemed to have a significant shorter time to progress to acute myeloid leukemia [slow-agers (n = 3) = 803.00 ± 43.41 days; fast-agers (n = 2) = 137.50 ± 53.03 days]. Conclusion In our analysis of 101 LR-MDS patients, increased epigenetic age acceleration (EAA) was observed in almost all epigenetic clocks evaluated. IPSS-M scoring correlated with EAA, confirming the relevance of somatic mutations. Cluster analysis revealed 3 EAA distinct groups. Patients with the highest EAA, that we termed fast-agers, had lower levels of pro-inflammatory cytokines compared to slow-agers. Overall, EAA effectively identifies clusters of patients with distinct cytokine profiles, stressing the modulating role of inflammation (and genetic alterations) in the pathophysiology of LR-MDS.

Introduction Biallelic TP53 inactivation negatively affects overall survival (OS) and leukemia-free survival (LFS) of patients with myelodysplastic syndromes (MDS), whereas MDS patients with monoallelic TP53 defects display outcomes similar to those of TP53wt patients. Biallelic TP53 inactivation is defined by WHO 2022 classification as the presence of multiple TP53 mutations or evidence of TP53 copy number loss, like del(17p) and copy neutral loss of heterozygosity (cnLOH). However, detection of del(17p) and cnLOH requires advanced cytogenetic evaluations, not routinely available in all centers. Both WHO and ICC 2022 classifications indicate variant allele frequency (VAF) ≥50% as a surrogate for definition of multi-hit status. Methods MDS patients (n=2496) from the publicly available dataset of the IPSS-M study by E. Bernard et al. were analyzed. An ROC curve was used to identify an optimal TP53 VAF threshold to discriminate mono- from multi-hit status. Cox proportional hazards modeling with stepwise variable selection was adopted to correlate VAF threshold with OS and LFS and to compare it with the conventional 50% VAF. Results Our analysis showed that a TP53 VAF ≥24% adequately predicted multi-hit status (AUC=0.81, sensitivity=0.81, specificity=0.74) and the Cox proportional hazards model confirmed the 24% VAF threshold as a stronger predictor for OS and LFS than the 50% threshold. Additionally, MDS patients with TP53 VAF ≥24% had similar outcomes to MDS patients with confirmed TP53 multi-hit status (median OS 0.77 and 0.76 years, LFS 0.65 and 0.64 years, respectively). Further supporting the threshold, patients with TP53 VAF <24% and single-hit TP53 displayed similar OS (2.52 vs 2.24) and LFS (2.2 vs 2.2 years). Thus, the TP53 24% VAF threshold seem to exhibits the same prognostic power as TP53 allelic status for OS and LFS across distinct IPSS-R categories, achieving statistical significance in all but low- and high-risk categories. Conclusion Our results showed that a TP53 VAF threshold of 24% informs MDS prognosis equally to TP53 allelic status. This parameter offers a simpler approach in the clinical setting in centers where advanced diagnostics are not available.

MDS.Following US Food and Drug Administration breakthrough designation for IVO in this population, enrollment was expanded.Objective: Report updated safety/efficacy expanded cohort results.Participants: Aged ≥18 years with mIDH1 R/R MDS following standard-of-care therapy.Interventions: Oral once daily IVO 500 mg in 28-day cycles.Outcomes: Safety/efficacy endpoints as of 26 September 2022.Results: Median (range) age: 73.0 (52-82) years, 78.9% patients had experienced HMA failure, median IDH1 variant allele frequency: 19.7% (n=19).Treatment-related adverse events occurred in 8 (42.1%) patients; grade 1 QTc interval increase in 1 (5.3%) and grade 2 differentiation syndrome in 2 (10.5%); none led to IVO discontinuation.In the efficacy population (n=18), ORR was 83.3% (95% CI: 58.6-96.4).Best overall responses were: CR, 7 (38.9%;95% CI: 17.3-64.3);mCR, 8 (44.4%; 95% CI: 21.5-69.2; 4 had hematologic improvement in ≥1 lineage); stable disease, 2 (11.1%; 95% CI: 1.4-34.7);and progressive disease, 1 (5.6%; 95% CI: 0.1-27.3).Median (range) time to CR: 1.87 (1.0-5.6)months; median CR duration: not reached.Median (range) overall survival duration: 35.7 (3.7-88.7)months.Of patients requiring transfusion at baseline, 5/7 (71.4%) became independent to red blood cell (RBC) transfusion and 3/4 (75.0%) to platelet transfusion.Of those transfusion independent at baseline, 9/11 (81.8%) patients remained independent from RBCs and 14/14 (100%) from platelets.At data cut-off, 12 (63.2%)patients were alive; 2 (11.1%) had received stem cell transplant and 2 (11.1%) had progressed to acute myeloid leukemia.Conclusion: IVO demonstrated an acceptable safety profile with durable remissions in a substantial proportion of patients with mIDH1 R/R MDS.Transfusion independence was achieved or maintained in most patients.

Vaginal dysbiosis is characterized by a decrease in the relative abundance of Lactobacillus species in favor of other species. This condition facilitates infections by sexually transmitted pathogens including high risk (HR)-human papilloma viruses (HPVs) involved in the development of cervical cancer. Some vaginal dysbiosis bacteria contribute to the neoplastic progression by inducing chronic inflammation and directly activating molecular pathways involved in carcinogenesis. In this study, SiHa cells, an HPV-16-transformed epithelial cell line, were exposed to different representative vaginal microbial communities. The expression of the HPV oncogenes E6 and E7 and the production of relative oncoproteins was evaluated. The results showed that Lactobacillus crispatus and Lactobacillus gasseri modulated the basal expression of the E6 and E7 genes of SiHa cells and the production of the E6 and E7 oncoproteins. Vaginal dysbiosis bacteria had contrasting effects on E6/E7 gene expression and protein production. The expression of the E6 and E7 genes and the production of the relative oncoproteins was increased by strains of Gardnerella vaginalis and, to a lesser extent, by Megasphaera micronuciformis. In contrast, Prevotella bivia decreased the expression of oncogenes and the production of the E7 protein. A decreased amount of p53 and pRb was found in the cultures of SiHa cells with M. micronuciformis, and accordingly, in the same cultures, a higher percentage of cells progressed to the S-phase of the cell cycle compared to the untreated or Lactobacillus-stimulated cultures. These data confirm that L. crispatus represents the most protective component of the vaginal microbiota against neoplastic progression of HR-HPV infected cells, while M. micronuciformis and, to a lesser extent, G. vaginalis may directly interfere in the oncogenic process, inducing or maintaining the production of viral oncoproteins.

Three and a half years after the pandemic outbreak, now that WHO has formally declared that the emergency is over, COVID-19 is still a significant global issue. Here, we focus on recent developments in genetic and genomic research on COVID-19, and we give an outlook on state-of-the-art therapeutical approaches, as the pandemic is gradually transitioning to an endemic situation. The sequencing and characterization of rare alleles in different populations has made it possible to identify numerous genes that affect either susceptibility to COVID-19 or the severity of the disease. These findings provide a beginning to new avenues and pan-ethnic therapeutic approaches, as well as to potential genetic screening protocols. The causative virus, SARS-CoV-2, is still in the spotlight, but novel threatening virus could appear anywhere at any time. Therefore, continued vigilance and further research is warranted. We also note emphatically that to prevent future pandemics and other world-wide health crises, it is imperative to capitalize on what we have learnt from COVID-19: specifically, regarding its origins, the world's response, and insufficient preparedness. This requires unprecedented international collaboration and timely data sharing for the coordination of effective response and the rapid implementation of containment measures.

Background Oral formulations ofhypomethylating agents decitabine (ASTX727) and azacitidine (cc-486, AZA) were approved respectively for treatment of higher risk myelodysplastic syndromes (HR-MDS) and for maintenance of acute myeloid leukemia after remission. Management of MDS patients with oral agents is relevant to improve quality of life. Oral-AZA has shown activity in lower risk MDS and its use is under evaluation in that setting. There are no data regarding maintenance of response achieved by subcutaneous (sc) AZA in HR-MDS cases by switching to the oral formulation. Aim The primary objective of the study was to explore the feasibility of replacing sc-AZA by its oral formulation in patients with HR-MDS in response. Maintenance or improvement of response and, as secondary objectives, patient reported outcome (PRO) and DNA methylation pattern were evaluated. Methods A monocentric, pilot phase 2 study was planned to enroll 11 subjects with confirmed diagnosis of MDS, IPSS-R higher risks, aged ≥ 65 years, in CR/CRi, PR or SD with HI after > 6 cycles of sc-AZA therapy (ClinicalTrial.gov NCT04806906). Patients were to receive 300 mg oral-AZA for the first 14-days of each 28-day treatment cycle until absence of benefit or disease progression. Dose of oral-AZA could be de-escalated based on toxicity. At screening and every 4 cycles, IWG response was evaluated. NGS evaluation of somatic mutations and DNA methylation analysis were performed at the same time points. DNA methylation pattern of separated BM CD34 positive cells was determined by Oxford Nanopore. At day1 of every cycle the EQ-5D questionnaire for QoL was administered. Feasibility, safety, tolerability as well as efficacy of oral-AZA were evaluated by monitoring AEs and response/loss of response, time to treatment discontinuation, and PRO. Results. As per July 31 st 2023, 11 HR-MDS patients with a median follow up of 167 days (range 47-805) were enrolled in this study. Male/female ratio was 4.5/1 with a median age of 81 yrs (67-88). At diagnosis patients belonged to IPSS-R risk categories intermediate 1/11, high 8/11, very high 2/11; IPSS-M risk categories at start of oral AZA: 3/11 very high, 6/11 high and 2/11 very low. Median number of sc-AZA cycles was 9 (range 7-52), while for oral-AZA median number of cycles was 4 (range 1-10). Oral AZA dose of 300 mg/day was maintained for all patients. DNA methylation was determined with success at baseline and every 4 cycles for all treated patients, and methylation status analyzed. None of the patients had serious adverse events related to study drug. Myelosuppressive effects were transient and Grade1/2. Patients maintained CR (4/11), PR (6/11) and SD with HI (1/11) achieved with sc-AZA, until progression. Therapy was interrupted for progression in 4 patients who evolved to AML after a median of 9 cycles of treatment with oral-AZA. Early discontinuation was experienced in 2/11 patients: in one case following a GI event grade 3 and patient decision to avoid de-escalation of the dose for subsequent cycles, in the second case because of patient reduced compliance. Both patients re-switched from oral to sc-AZA treatment and are still in response (PR). PRO analyses indicated that oral-AZA therapy improved most quality-of-life domains compared to sc-AZA. Treatment is ongoing for 4/11 patients in CR (3/4) and PR (1/4). Responses were observed across all IPSS-M risk categories. Conclusions Treatment of HR-MDS elderly patients with oral-AZAis feasible and effective. We observed duration of hematological response with a length consistent with what shown in this patient subgroup, and even after a very prolonged treatment with sc-AZA. Oral AZA was generally well tolerated and AEs did not differ from those observed for sc-AZA.