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Objective Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS). Though MS was initially considered to be a white matter demyelinating disease, myelin loss in cortical gray matter has been reported in all disease stages. We previously identified microRNAs (miRNAs) in white matter lesions (WMLs) that are detected in serum from MS patients. However, miRNA expression profiles in gray matter lesions (GMLs) from progressive MS brains are understudied. Methods We used a combination of global miRNAs and gene expression profiling of GMLs and independent validation using real‐time quantitative polymerase chain reaction (RT‐qPCR), immuno‐in situ hybridization, and immunohistochemistry. Results Compared to matched myelinated gray matter (GM) regions, we identified 82 miRNAs in GMLs, of which 10 were significantly upregulated and 17 were significantly downregulated. Among these 82 miRNAs, 13 were also detected in serum and importantly were associated with brain atrophy in MS patients. The predicted target mRNAs of these miRNAs belonged to pathways associated with axonal guidance, TGF‐β signaling, and FOXO signaling. Further, using state‐of‐the‐art human protein–protein interactome network analysis, we mapped the four key GM atrophy‐associated miRNAs (hsa‐miR‐149*, hsa‐miR‐20a, hsa‐miR‐29c, and hsa‐miR‐25) to their target mRNAs that were also changed in GMLs. Interpretation Our study identifies miRNAs altered in GMLs in progressive MS brains that correlate with atrophy measures. As these miRNAs were also detected in sera of MS patients, these could act as markers of GML demyelination in MS.

Dyskeratosis congenita is a prototypic inherited telomeropathy. Telomere length (TL) shortening has also been described in aplastic anemia (AA) and attributed in occasional patients to the presence of mostly heterozygous germline alterations in telomerase machinery genes (most commonly in the reverse transcriptase gene, TERT). Among various heterozygous variants described, certain TERT variants associated with shortened TL (e.g. H412Y and A202T) showed later a higher prevalence in general population questioning their pathogenicity and role as risk alleles. Nevertheless, shortened TL has been shown to correlate with poor outcomes in AA and myelodysplastic syndromes (MDS) along with a higher progression rate to acute myeloid leukemia, and increased non-relapse mortality after HSCT. A high prevalence (41/1514; 2.7%) of non-recurrent germline TERT rare variants, has been found in patients with MDS undergoing HSCT (Reilly et al Blood 2021) without any clinically apparent diagnosis of a telomere biology disorder. Despite their classification as VUS, these alterations were shown to result in impairment of telomere elongation in vitro and reduced TL in vivo. Clinically, VUS carriers were also characterized by younger age at MDS presentation (median 52 years) and shorter survival due to a higher incidence of non-relapse mortality. We hypothesized that should such rare variants (traditionally thought as VUS) represent founder lesions and constitute risk alleles, one would expect to find some recurrence of these lesions in other cohorts of patients with myeloid neoplasia (MN). To test this hypothesis we screened a large meta-analytic cohort of MN patients (n=2560) including our patients (The Cleveland Clinic Foundation and MLL-Munich Leukemia laboratory) and a public series (The BEAT AML Master Trial and TCGA among others) for the presence of TERT coding variants. When whole genome sequencing was available (n=1432) TelomereHunter tool was used to estimate TL in carriers of TERT rare variants and wild-type (WT) cohorts. Overall, we identified 73 TERT coding variants. Based on maximum gnomAD population allele frequency (AF) In terms of clinical features, MN patients with TERT rare variants harbored more somatic hits in DNMT3A and SF3B1 (26% each), and FLT3 (15%) with a diverse spectrum of additional molecular lesions, including germline VUS of other genes (4 patients harbored concomitant VUS in BRCA1, FANCG, and SAMD9, with another patient carrying a likely benign SAMD9L variant) (Fig1). When compared to WT counterparts, patients harboring TERT rare variants had a similar median age at MN diagnosis (70 vs. 66, P= 0.38) and similar survival outcomes (P= 0.96). Importantly, HSCT outcomes were ascertained only in 7 cases because of the real-life nature of our cohort which compiles MN patients of any subtype and age, thereby not biased by transplant referral like in the aforementioned study focusing only on MDS patients undergone HSCT. Finally, principal causes of death were primary disease (63%), presence of another malignancy (19%) and infections (13%) and, of note, no patient died of non-infectious pulmonary causes. In sum, our study, while confirming a relatively high (albeit not significant vs. controls) combined TERT VUS population burden in MN, also strongly indicates that rare TERT VUS are non-recurrent. Therefore, they cannot be considered risk alleles individually nor can their biological (combined or individual) impact in terms of non-transplant outcomes, irrespective of their in vitro functional effects. Figure 1 Figure 1. Disclosures Carraway: Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Takeda: Other: Independent review committee; Astex: Other: Independent review committee; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy; Novartis: Consultancy; Regeneron: Consultancy.

Clinical impact and mechanistic contributions to leukemogenesis are difficult to assign to less common somatic mutations. However, the genetics of inherited syndromes can often be helpful in discerning the biological functions and mechanistic consequences of genes in other diseases. PHF6 (Xq26.2) encodes a protein consisting of two PHD-type zinc finger domains with activity in transcriptional regulation. PHF6 translocations were originally described in T-ALL and its mutations were later observed also in CML and adult AML. Germline (GL) PHF6MT cause Börjeson-Forssman-Lehmann syndrome (BFLS), an X-linked disorder characterized by intellectual disability and, to date, only a few BFLS cases were found to develop lymphoma or T-ALL. While regularly encountered in myeloid neoplasia (MN), the impact and functional meaning of PHF6 are not well established. To determine the incidence, distribution and molecular context of PHF6MT we studied a large cohort of patients with MN (n=8617) collected from our institution and public series. 1 Overall, 73% of patients were AML (pAML 69%; sAML 4%), MDS (22%) and MDS/MPN (5%) with a median age at diagnosis of 67 ys (18-100). We detected 149 patients (2%) carrying at least 1 PHF6MT with 11 harboring more than 1 hit. Four patients carried -X in addition to PHF6MT (2 males; 2 females). Majority of patients (68%) carried frameshift del/ins and nonsense. Mutations were scattered across all coding region with a slightly enrichment (47%) in the second PHD domain (239-330 aa) including the frequent R274Q/X (n=17). Common hits mainly affected arginine residues essential for DNA binding capacity (R129X n=9, R116X=7, R319X=5, R225X=3) followed by other hits (I314T=6, Y301X and C20fs=4 each). Of note, R116X, R225X, R274X, C280Y, H329R and Y303* lesions overlapped with the T-ALL PHF6MT spectrum while no overlap was found with GL mutations found in BFLS. Overall, 75% of all PHF6MT carriers were males and carried mostly (80%) truncating lesions. Compared to mutational frequencies observed in other X-linked genes, truncating PHF6MT behaved similarly to those in ZRSR2 (78%), STAG2 (73%) and BCOR (62%). Conversely, BCORL1MT, KDM6AMT and PIGAMT were evenly distributed between genders. When evaluating mutational characteristics in males and females, no differences were found in sex-adjusted median variant allelic burden of PHF6MT (54.8 vs 51%) nor its mRNA expression levels suggesting locus inactivation. PHF6MT tended to be older than PHF6WT patients (72 vs 68 ys; P= .05) and had mostly (63%) AML followed by MDS (23%) and MDS/MPN (14%). OS was similar between PHF6MT and PHF6WT patients (P= .16). Expression analyses showed that PHF6 loss leads to deregulation of chromatin and transcriptional factor genes. Indeed, in our cohort the most comutated genes were transcriptional factors and chromatin modifiers genes such as RUNX1 (26/149, 17%), ASXL1 (23/149, 15%) and TET2 (17/149, 11%). Of note, this group characterized by the triple ASXL1, RUNX1, TET2 mutational configuration clustered in one of the genomic groups previously identified (GC-3) 1 but the presence of these lesions did not worsen the OS as compared to PHF6MT without this mutational constellation. A low frequency of SF3B1MT (4%) was also noted confirming the enrichment of PHF6MT in AML rather than in low risk MDS. Further, 12% (14 males; 4 females) of PHF6MT patients had X-mutation mosaicism as shown by concomitant hits in BCOR (n=8), ZRSR2 (4), STAG2 (5), KDM6A (1). PHF6MT were equally founder lesions (30%; 44/149) and subclonal (34%; 50/149) whereas the rest was indistinguishable by VAF discrimination (co-dominant). The most common subclonal mutations were U2AF1 (14%, 6/44), IDH1/2 (9%, 4/44) and RUNX1 (7%, 3/44). When PHF6MT were subclonal, the founder hits were in TET2 (14%, 7/50), DNMT3A and RUNX1 (12%, each 6/50) genes. Given the high frequency of RUNX1MT in PHF6MT we investigated whether RUNX1 and PHF6 might be correlated. Transcriptomic analysis of 6246 patients (from 9 public studies) 2 showed a direct linear correlation (AdjR2= .03, P=5.55e-05) between the expression of the two genes. Our study is the largest to date to investigate the genetic landscape of PHF6MT in MN and highlights a strong connection of PHF6 with transcriptional regulation and chromatin genes. Ongoing scDNA-seq will clarify whether these mutations were acquired in distinct clones helping in dissecting the clonal hierarchy of PHF6MT cases. Disclosures Carraway: Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Takeda: Other: Independent review committee; Astex: Other: Independent review committee; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Advani: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Glycomimetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Macrogenics: Research Funding; Immunogen: Research Funding; OBI: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Research Funding. Maciejewski: Regeneron: Consultancy; Novartis: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.

Structural and functional variability of human leukocyte antigen (HLA) is the foundation for competent anti-tumor and infectious adaptive immune responses. HLA genomic heterogeneity enables the presentation of a broad immune-peptidome, sustaining an efficient diversification of T cell receptor repertoires (TCR). 1,2,3 Any perturbation impacting this diversity may be at the basis of pathological processes, hampering antigen presentation capabilities and T-cell reactivity. In allogeneic hematopoietic cell transplantation (allo-HCT) setting, the graft versus leukemia (GvL) effect should ensure disease control allowing the recognition of recipient neoantigen burden by donor T-cell effectors. However, the molecular dissection of graft versus host responses (GvH) remains elusive. Herein, by means of a broad immunogenetic study of a cohort of patients with myeloid malignancies who received a donor matched allo-HCT, we investigated how dysfunction of HLA variability could have an impact on alloreactive responses, ultimately hindering disease control. To that end, we combined NGS-based HLA genotyping and TCR-beta sequencing to molecularly characterize the HLA region in terms of locus-specific divergence and somatic mutational profile, and dissect features and clonotypic spectra of TCR repertoires. We first hypothesized that more diverse HLA genotypes could better present leukemic neoantigen burden than less diverse complexes, enhancing the GvL effect. Hence, we performed a matched-pair analysis between allo-HCT recipients relapsing after 3mo (median 6.2 mo. [IQR=4.6-12]), N=75) compared to patients without recurrence (N=193, matched for ethnicity, age, disease, graft source and conditioning regimens) and characterized the patterns of HLA evolutionary divergence (HED), 1 a metric recently conceived to quantitate the pair-wise distance (based on physiochemical composition) between the amino acids located within the peptide-binding groove of two homologous HLA alleles. Overall, the relapsed group was characterized by a lower global (class I/II) mean HED (p=.0029) compared to non-relapsed patients, with major differences seen for C (p=.0041), DQB1 (p=.0291), and DPB1 (p=.0396) loci. When studying the landscape of post-transplant TCR reconstitution (+3 months) in a subset of 25 patients, we observed an inverse correlation between TCR clonal expansion and global HED (AdjR 2=0.04, p= Altogether these findings pinpoint the role of the dysfunction of the structural variability of HLA complexes within both germline (HED) and somatic (HLA loss/mutations) scenarios as mechanisms hampering a successful neoantigen presentation and TCR recovery processes, possibly conveying a higher risk of disease relapse or treatment-resistance. Disclosures Balasubramanian: Servier Pharmaceuticals: Research Funding. Carraway: Takeda: Other: Independent review committee; AbbVie: Other: Independent review committee; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astex: Other: Independent review committee. Hamilton: Syndax: Membership on an entity's Board of Directors or advisory committees; Equilium: Membership on an entity's Board of Directors or advisory committees. Majhail: Anthem, Inc: Consultancy; Incyte Corporation: Consultancy. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Regeneron: Consultancy; Alexion: Consultancy; Novartis: Consultancy.