Patient demographic data for the female patients in the Extragonadal GCT cohort (both Make-an-IMPACT and internal MSK patients, n=16)

Omidubicel-onlv is an FDA-approved, nicotinamide-modified, allogeneic hematopoietic progenitor cell therapy derived from umbilical cord blood (UCB). A phase 3 study demonstrated improved hematopoietic recovery and decreased infections with omidubicel compared with UCB allogeneic transplantation. We report results of an Expanded Access Program evaluating clinical outcomes in patients with hematologic malignancies following transplantation with omidubicel. Between August 2020 and May 2023, 29 patients were transplanted at 5 US sites. Patients received myeloablative conditioning, prophylactic and therapeutic medications, and supportive care per institutional guidelines, and were monitored for engraftment, infections, and graft-versus-host-disease (GVHD) for up to 2 years post-transplant. Results were compared with previously reported phase 3 outcomes. Omidubicel recipients had a median age of 39 (range 20-73, 62% male); 45% were non-White and 65.5% had acute leukemia. Median follow-up was 11.8 (range: .3-27.7) months. Median neutrophil and platelet engraftment times were 12 and 33.5 days, respectively. Acute GVHD (grade 3-4) at day 100 occurred in 19% of patients, with chronic GVHD at 1 year in 9% of patients, all of which were mild. First grade 2 to 3 bacterial infections through 100 days post-transplant and first grade 3 viral infection 1 year post-transplant occurred in 18% and 12% of patients, respectively. One-year disease-free survival and overall survival rates were 76% and 87%, respectively. This real-world study of omidubicel transplantation for hematologic malignancies finds that this graft source is commonly used for non-White allogeneic transplant recipients. The rapid engraftment kinetics observed following transplantation with omidubicel appears to have addressed excessive nonrelapse mortality that has been previously observed following myeloablative umbilical cord blood transplantation.

Genomically matched targeted therapies guided by MSK-IMPACT testing (n=22).

Clonality assessment of known oncogenic mutations in the female GCT cohort.

SummaryHistiocytic neoplasms are diverse clonal haematopoietic disorders, and clinical disease is mediated by tumorous infiltration as well as uncontrolled systemic inflammation. Individual subtypes include Langerhans cell histiocytosis (LCH), Rosai–Dorfman–Destombes disease (RDD) and Erdheim–Chester disease (ECD), and these have been characterized with respect to clinical phenotypes, driver mutations and treatment paradigms. Less is known about patients with mixed histiocytic neoplasms (MXH), that is two or more coexisting disorders. This international collaboration examined patients with biopsy‐proven MXH with respect to component disease subtypes, oncogenic driver mutations and responses to conventional (chemotherapeutic or immunosuppressive) versus targeted (BRAF or MEK inhibitor) therapies. Twenty‐seven patients were studied with ECD/LCH (19/27), ECD/RDD (6/27), RDD/LCH (1/27) and ECD/RDD/LCH (1/27). Mutations previously undescribed in MXH were identified, including KRAS, MAP2K2, MAPK3, non‐V600‐BRAF, RAF1 and a BICD2‐BRAF fusion. A repeated‐measure generalized estimating equation demonstrated that targeted treatment was statistically significantly (1) more likely to result in a complete response (CR), partial response (PR) or stable disease (SD) (odds ratio [OR]: 17.34, 95% CI: 2.19–137.00, p = 0.007), and (2) less likely to result in progression (OR: 0.08, 95% CI: 0.03–0.23, p < 0.0001). Histiocytic neoplasms represent an entity with underappreciated clinical and molecular diversity, poor responsiveness to conventional therapy and exquisite sensitivity to targeted therapy.

Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 4226 Hierarchical clustering on principal components subdivided the ECD patient sample into clusters which reflect what has often already been observed in the clinical practice, with regard to the widespread disease phenotype as opposed to the limited one, and with regard to the phenotypes of ECD associated with other types of histiocytosis or myelodysplastic syndrome. HemaSphere | 2023;7(S3) EHA2023 Hybrid Congress Copyright Information: (Online) ISSN: 2572-9241 © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association. This is an open access Abstract Book distributed under the Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) which allows third parties to download the articles and share them with others as long as they credit the author and the Abstract Book, but they cannot change the content in any way or use them commercially. Abstract Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx.Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 4227

Background: Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm characterized by the accumulation of clonal dendritic cells expressing CD1a and CD207 (Langerin). LCH affects both children and adults and typically ranges from an indolent unifocal form to a progressive multisystemic disease. In the past decade, in depth molecular profiling of LCH as well as other histiocytic neoplasms demonstrated that these diseases are fundamentally dependent on somatic MAPK activating mutations, with the canonical V600E BRAF mutation emerging in more than 50% of LCH patients. Nonetheless, MAPK independent driver mutations were previously reported and anecdotal evidence of successful targeted treatment of various histiocytoses harboring non-canonical drivers such as RET, ALK and CSF1R were previously described in the literature. Aims: To report a case of PI3K driven LCH treated with an isoform specific PIK3CA inhibitor. Methods: Case summary: a 46 year-old mother of 3 presented with complaints of progressive headaches, polydipsia and polyuria. Following a pathological water deprivation test and identification of pituitary stalk thickening on MRI, a diagnosis of central diabetes insipidus (CDI) was established and desmopressin treatment was initiated. However, 10 months following her CDI diagnosis, the patient began to complain of sub febrile episodes of fever and worsening dry cough. Imaging studies including PET/CT disclosed extensive bilateral reticulo-nodular infiltration, nodules with cystic transformation and ground glass opacity, suspicious of pulmonary LCH. Histological examination of a wedge biopsy specimen obtained from the lungs confirmed the diagnosis of LCH and consequent molecular studies identified the M1043V PIK3CA mutation. During her medical investigation, the patient’s conditioned worsened, with new disease foci emerging – including cervical lymphadenopathy and a lytic vertebral lesion involving D11. The patient then received palliative radiotherapy directed at the D11 lytic lesion followed by escalating systemic therapy with single agent Alpelisib, via the Managed Access Program at Novartis. Response to treatment was assessed clinically and using PET/CT. At the molecular level, the effect of Alpelisib was assessed by measuring PTEN expression levels and cell cycle regulating non-coding RNA molecules by using quantitative real-time polymerase chain reaction (qRT-PCR). Results: Treatment with Alpelisib resulted in rapid amelioration of night sweats within 6 days, decrease in back pain within 3 weeks and normalization of all abnormally FDG avid disease foci within 3 months of treatment. The complete metabolic response observed persists currently more than a year following treatment initiation. Treatment with Alpelisib is well tolerated except for a mild increase in HbA1C levels to 6.7%. Moreover, PTEN, the main regulator of the PI3K pathway was found to be upregulated following 3 and 9 months of treatment, in parallel to downregulation of small non-coding RNAs molecules that regulates its expression. Image:Summary/Conclusion: This is the first study demonstrating that the alpha catalytic subunit of PI3K is a targetable non-canonical driver of LCH.

Introduction Omidubicel-onlv is a nicotinamide-modified allogeneic hematopoietic progenitor cell therapy donor source derived from umbilical cord blood (UCB). A phase 3 randomized study (NCT02730299) demonstrated improved hematopoietic recovery and decreased infections in patients transplanted with omidubicel compared to UCB. An expanded access program (EAP) was conducted to provide access to omidubicel in advance of commercial availability in the United States (US) in April 2023. Objective This was an open-label, single-arm EAP evaluating clinical outcomes in patients with hematologic malignancies following allo-HCT with omidubicel. Methods Eligible patients (≥12 years) with hematologic malignancies received myeloablative conditioning, prophylactic medications, and supportive care as per institutional guidelines. Patients were monitored for neutrophil and platelet engraftment, infections, graft versus host disease (GVHD), and additional clinical events for up to 2 years post-transplantation. Results were retrospectively compared with previously described outcomes in patients from the phase 3 study comparing transplant with omidubicel (P3-OMI, n=52) and unmanipulated UCB (P3-UCB, n=56). Results Between July 2020 and April 2023, 36 patients were enrolled and 29 (18 males, 11 females) were transplanted at 5 US sites. Median age was 39 years (range: 20-73); 55% were White, 21% Asian, 17% Black, and 7% other. Diagnoses were acute myelogenous leukemia (38%), acute lymphocytic leukemia (28%), myelodysplastic syndrome (21%), and other (14%). All patients received myeloablative conditioning (total body irradiation-based: 58.5%, chemotherapy alone: 34.5%). Median transplanted CD34+ cell dose was 5.6x10 6 cells/kg (range: 1.8-18.7x10 6 cells/kg) and HLA match was 4/6 in 52%, 5/6 in 45%, and 6/6 in 3%. Median follow-up was 6.3 months (range: 0.3-27.7). No infusion reactions were reported. Median time to neutrophil (≥500/µl x3 days) and platelet (>20,000/µl x3 days) engraftment were 13 and 34 days, respectively. Primary graft failure occurred in 2 (6.9%) patients, both alive >100 days after second haploidentical transplant. The incidences of first grade 2-3 bacterial and invasive (grade 3) fungal infection through 100 days post-transplant were 16.9% and 0%, respectively. The incidence of grade 3 viral infections 1-year post-transplant was 13.4%. At 100 and 365 days post-transplant, disease-free survival (DFS) rates were 95.2% and 82.5%, and overall survival (OS) rates were 96.3% and 84.2%, respectively. Acute GVHD was reported in 13 (44.8%) patients (grade 2: 34.5%, grade 3: 10.3%) and mild chronic GVHD in 1 (3.5%) patient. There were 4 deaths (13.8%): 2 due to relapse (Days 275 and 381), 1 due to infection (Day 135) and 1 due to respiratory failure (Day 10). Post-transplant lymphoproliferative disease was reported in 1 patient. Demographic and disease characteristics were similar to those in the phase 3 omidubicel registration trial, including racial and ethnic diversity (>39% minorities). Neutrophil engraftment time in EAP patients was similar to P3-OMI (10 days) and shorter than P3-UCB (20.5 days). Similarly, median platelet recovery was similar to P3-OMI (37 days) and shorter than P3-UCB (50 days). Infection, GVHD, DFS, and OS in EAP were also similar to those observed in P3-OMI (Table 1). Conclusion In a real-world EAP setting, the outcomes of allo-HCT with omidubicel in patients with hematologic malignancies were consistent with those from the phase 3 registration study. These data support the role of omidubicel as a donor source, particularly for patients from diverse racial backgrounds.

BACKGROUND:Ovarian cancer (OC) is known for exhibiting low response rates to immune checkpoint inhibitors that activate T cells. However, immunotherapies that activate B cells have not yet been extensively explored and may be a potential target, as B cells that secrete immunoglobulins have been associated with better outcomes in OC. Although the secretion of immunoglobulins is often mediated by the microbiome, it is still unclear what role they play in limiting the progression of OC.METHODS:We conducted an in-vivo CRISPR screen of immunodeficient (NSG) and immune-intact wild type (WT) C57/BL6 mice to identify tumor-derived immune-escape mechanisms in a BRAC1- and TP53-deficient murine ID8 OC cell line (designated ITB1). To confirm gene expression and signaling pathway activation in ITB1 cells, we employed western blot, qPCR, immunofluorescent staining, and flow cytometry. Flow cytometry was also used to identify immune cell populations in the peritoneum of ITB1-bearing mice. To determine the presence of IgA-coated bacteria in the peritoneum of ITB1-bearing mice and the ascites of OC patients, we employed 16S sequencing. Testing for differences was done by using Deseq2 test and two-way ANOVA test. Sequence variants (ASVs) were produced in Qiime2 and analyzed by microeco and phyloseq R packages.RESULTS:We identified tumor necrosis factor receptor-associated factor 3 (TRAF3) as a tumor-derived immune suppressive mediator in ITB1 cells. Knockout of TRAF3 (TRAF3KO) activated the type-I interferon pathway and increased MHC-I expression. TRAF3KO tumors exhibited a growth delay in WT mice vs. NSG mice, which was correlated with increased B cell infiltration and activation compared to ITB1 tumors. B cells were found to be involved in the progression of TRAF3KO tumors, and B-cell surface-bound and secreted IgA levels were significantly higher in the ascites of TRAF3KO tumors compared to ITB1. The presence of commensal microbiota was necessary for B-cell activation and for delaying the progression of TRAF3KO tumors in WT mice. Lastly, we observed unique profiles of IgA-coated bacteria in the ascites of OC-bearing mice or the ascites of OC patients.CONCLUSIONS:TRAF3 is a tumor-derived immune-suppressive modulator that influences B-cell infiltration and activation, making it a potential target for enhancing anti-tumor B-cell responses in OC.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm characterized by the accumulation of clonal mononuclear phagocyte system cells expressing CD1a and CD207. In the past decade, molecular profiling of LCH as well as other histiocytic neoplasms demonstrated that these diseases are driven by MAPK activating alterations, with somatic BRAFV600E mutations in >50% of patients with LCH, and clinical inhibition of MAPK signaling has demonstrated remarkable clinical efficacy. At the same time, activating alterations in kinase-encoding genes, such as PIK3CA, ALK, RET, and CSF1R, which can activate mitogenic pathways independent from the MAPK pathway, have been reported in a subset of histiocytic neoplasms with anecdotal evidence of successful targeted treatment of histiocytoses harboring driver alterations in RET, ALK, and CSF1R. However, evidence supporting the biological consequences of expression of PIK3CA mutations in hematopoietic cells has been lacking, and whether targeted inhibition of PI3K is clinically efficacious in histiocytic neoplasms is unknown. Here, we provide evidence that activating mutations in PIK3CA can drive histiocytic neoplasms in vivo using a conditional knockin mouse expressing mutant PIK3CAH1047R in monocyte/dendritic cell progenitors. In parallel, we demonstrate successful treatment of PIK3CA-mutated, multisystemic LCH using alpelisib, an inhibitor of the alpha catalytic subunit of PI3K. Alpelisib demonstrated a tolerable safety profile at a dose of 750 mg per week and clinical and metabolic complete remission in a patient with PIK3CA-mutated LCH. These data demonstrate PIK3CA as a targetable noncanonical driver of LCH and underscore the importance of mutational analysis-based personalized treatment in histiocytic neoplasms.

Abstract Purpose: To overcome barriers to genomic testing for patients with rare cancers, we initiated a program to offer free clinical tumor genomic testing worldwide to patients with select rare cancer subtypes. Experimental Design: Patients were recruited through social media outreach and engagement with disease-specific advocacy groups, with a focus on patients with histiocytosis, germ cell tumors (GCT), and pediatric cancers. Tumors were analyzed using the MSK-IMPACT next-generation sequencing assay with the return of results to patients and their local physicians. Whole-exome recapture was performed for female patients with GCTs to define the genomic landscape of this rare cancer subtype. Results: A total of 333 patients were enrolled, and tumor tissue was received for 288 (86.4%), with 250 (86.8%) having tumor DNA of sufficient quality for MSK-IMPACT testing. Eighteen patients with histiocytosis have received genomically guided therapy to date, of whom 17 (94%) have had clinical benefit with a mean treatment duration of 21.7 months (range, 6–40+). Whole-exome sequencing of ovarian GCTs identified a subset with haploid genotypes, a phenotype rarely observed in other cancer types. Actionable genomic alterations were rare in ovarian GCT (28%); however, 2 patients with ovarian GCTs with squamous transformation had high tumor mutational burden, one of whom had a complete response to pembrolizumab. Conclusions: Direct-to-patient outreach can facilitate the assembly of cohorts of rare cancers of sufficient size to define their genomic landscape. By profiling tumors in a clinical laboratory, results could be reported to patients and their local physicians to guide treatment. See related commentary by Desai and Subbiah, p. 2339

Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis that frequently infiltrates the peri-kidney space ("hairy kidney" appearance), kidney pelvis and proximal ureters, leading to obstructive uropathy. Here, we analyzed the clinical characteristics, imaging findings and long-term kidney outcome of a large multicenter cohort comprising 195 consecutive patients with ECD. Retroperitoneal peri-kidney or peri-ureteral involvement was detected at diagnosis in 147 patients. Of them, 70 had hydronephrosis (bilateral in 47), and 16 with kidney atrophy (unilateral in 14). Kidney vascular peduncle infiltration was found in 60 patients, and kidney artery stenosis in 31. The estimated glomerular filtration rate (eGFR) at diagnosis was significantly lower in patients with than in those without peri-kidney involvement (median 74 vs. 98 mL/min/1.73 m2). Ureteral stenting often failed to achieve kidney function recovery. A total of 181 patients received medical therapies: first-line treatments included interferon-α (61%), BRAF-inhibitors (17%), mTOR-inhibitors (7%), or other drugs (15%). These therapies were efficacious for ECD but rarely induced kidney function improvement (one-year eGFR increase over 25% in under 10% of patients). After a median of 43 months, 19% of patients died and 5% developed kidney failure. Among patients with peri-kidney involvement, 44% developed chronic kidney disease (CKD) 3-5 at five years vs. 5% of those without. Unadjusted predictors of advanced CKD and kidney failure/death were age over 50 years, hypertension, BRAFV600E mutation, and baseline eGFR. At multivariable analysis, cardiovascular comorbidities were associated with advanced CKD, and age over 50 years with kidney failure/death. Thus, kidney involvement is common in ECD and can lead to CKD or kidney failure despite effective medical therapies or urological procedures.

Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative treatment for hematologic malignancies and nonmalignant disorders. Rapid immune reconstitution (IR) following allogeneic HCT has been shown to be associ-ated with improved clinical outcomes and lower infection rates. A global phase 3 trial (ClinicalTrials.gov NCT02730299) of omidubicel, an advanced cell therapy manufactured from an appropriately HLA-matched single umbilical cord blood (UCB) unit, showed faster hematopoietic recovery, reduced rates of infection, and shorter hospi-talizations in patients randomized to omidubicel compared with those randomized to standard UCB. This optional, prospective substudy of the global phase 3 trial characterized the IR kinetics following HCT with omidubicel compared with UCB in a systematic and detailed manner. This substudy included 37 patients from 14 global sites (omidubicel, n = 17; UCB, n = 20). Peripheral blood samples were collected at 10 predefined time points from 7 to 365 days post-HCT. Flow cytometry immunophenotyping, T cell receptor excision circle quantification, and T cell receptor sequenc-ing were used to evaluate the longitudinal IR kinetics post-transplantation and their association with clinical out-comes. Patient characteristics in the 2 comparator cohorts were overall statistically similar except for age and total body irradiation (TBI)-based conditioning regimens. The median patient age was 30 years (range, 13 to 62 years) for recipients of omidubicel and 43 years (range, 19 to 55 years) for UCB recipients. A TBI-based conditioning regimen was used in 47% of omidubicel recipients and in 70% of UCB recipients. Graft characteristics differed in their cellular composition. Omidubicel recipients received a 33-fold higher median dose of CD34+ stem cells and one-third of the median CD3+ lymphocyte dose infused to UCB recipients. Compared with UCB recipients, omidubicel recipients exhib-ited faster IR of all measured lymphoid and myelomonocytic subpopulations, predominantly in the first 14 days post-transplantation. This effect involved circulating natural killer (NK) cells, helper T (Th) cells, monocytes, and dendritic cells, with superior long-term B cell recovery from day +28. At 1 week post-HCT, omidubicel recipients exhibited 4.1 -and 7.7-fold increases in the median Th cell and NK cell counts, respectively, compared to UCB recipients. By 3 weeks post-HCT, omidubicel recipients were 3-fold more likely to achieve clinically relevant Th cell and NK cell counts >= 100 cells/AL. Similar to UCB, omidubicel yielded a balanced cellular subpopulation composition and diverse T cell receptor repertoire in both the short term and the long term. Omidubicels CD34+ cell content correlated with faster IR by day +7 post-HCT, which in turn coincided with earlier hematopoietic recovery. Finally, early NK and Th cell reconstitution correlated with a decreased rate of post-HCT viral infections, suggesting a plausible explanation for this phenomenon among omidubicel recipients in the phase 3 study. Our findings suggest that omidubicel efficiently promotes IR across multiple immune cells, including CD4+ T cells, B cells, NK cells, and dendritic cell subtypes as early as 7 days post transplantation, potentially endowing recipients of omidubicel with early protective immunity. (c) 2023 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Background: Histiocytic neoplasms are driven by mutations activating the MAPK/ERK pathway; however, their epigenetic landscape remains poorly understood. MicroRNAs (miRNAs) are epigenetic regulators implicated in cancer development. Moreover, DNA methylation of miRNAs affects hematological malignancies development. Our previous study identified a unique miRNA expression signature in Erdheim-Chester Disease (ECD) patients. To further understand the biological mechanisms of ECD, we investigated the involvement of miRNAs, genes, and their methylation status in ECD, Langerhans cell histiocytosis (LCH), and Rosai-Dorfman disease (RDD). Methods: Methylation profiles of 14 LCH, 6 ECD, and 10 RDD patients were determined from histyocitic infiltrated tissues (Illumina EPIC-methylation array). These were compared to controls, suture granulomas (n=4), localized inflammatory reactions characterized by non-neoplastic histiocytic infiltrates. miRNAome data was identified by NanoString analysis and qRT-PCR. Results: We identified significant alterations in 23,322 methylation sites (FDR<0.05, |Δß|≧20%) between controls and the histiocytosis group, indicating differential epigenetic regulation. ECD and LCH exhibited a shared methylation signature distinct from RDD. Pathway analyses revealed that the differentially methylated genes (DMGs) were involved in inflammatory response, RAS, TNF, and PI3K-mTOR signaling pathways, linked to histiocytic neoplasms. Specifically, we found DMG involvement in regulation and generation of non-coding RNAs. Notably, we observed significant downregulation of the let7-miRNA family, particularly let7b, in ECD and LCH patients. Subsequent analysis revealed hypermethylation of specific CpG islands in the let7b DNA sequence, potentially silencing let7b expression in these diseases. Given the let7-miRNA family's role in regulating the MAPK pathway, this suggests a novel mechanism in the pathogenesis of these neoplasms. Conclusions: We provide valuable insights into differential methylation patterns in histiocytosis neoplasms, highlighting the involvement of epigenetic modifications in their development and progression. This enhances our understanding of histiocytosis development and may contribute to the identification of novel diagnostic and therapeutic approaches for these patients.

The tumor microenvironment hosts antibody-secreting cells (ASCs) associated with a favorable prognosis in several types of cancer. Patient-derived antibodies have diagnostic and therapeutic potential; yet, it remains unclear how antibodies gain autoreactivity and target tumors. Here, we found that somatic hypermutations (SHMs) promote antibody antitumor reactivity against surface autoantigens in high-grade serous ovarian carcinoma (HGSOC). Patient-derived tumor cells were frequently coated with IgGs. Intratumoral ASCs in HGSOC were both mutated and clonally expanded and produced tumor-reactive antibodies that targeted MMP14, which is abundantly expressed on the tumor cell surface. The reversion of monoclonal antibodies to their germline configuration revealed two types of classes: one dependent on SHMs for tumor binding and a second with germline-encoded autoreactivity. Thus, tumor-reactive autoantibodies are either naturally occurring or evolve through an antigen-driven selection process. These findings highlight the origin and potential applicability of autoantibodies directed at surface antigens for tumor targeting in cancer patients.

Erdheim-Chester disease (ECD) is characterized by excessive production and accumulation of histiocytes within multiple tissues and organs. ECD patients harbor recurrent mutations of genes associated with the RAS/RAF/MEK/ERK signaling pathway, particularly, the BRAFV600E mutation. Following our previous finding that miR-15a-5p is the most prominently downregulated microRNA in ECD patients compared to healthy individuals, we elucidated its role in ECD pathogenesis. Bioinformatics analysis followed by a luciferase assay showed that chemokine ligand 10 (CXCL10) is a target gene regulated by miRNA-15a-5p. This was confirmed in 24/34 ECD patients that had low expression of miR-15a-5p concurrent with upregulated CXCL10. Overexpression of miR-15a-5p in cell lines harboring BRAF or RAS mutations (Ba/F3, KG-1a and OCI-AML3) resulted in CXCL10 downregulation, followed by LIN28a and p-ERK signaling downregulation and let-7 family upregulation. Overexpression of miR-15a-5p inhibited cell growth and induced apoptosis by decreasing Bcl-2 and Bcl-xl levels. Analysis of sequential samples from 7 ECD patients treated with MAPK inhibitors (vemurafenib/cobimetinib) for 4 months showed miR-15a-5p upregulation and CXCL10 downregulation. Our findings suggest that miR-15a-5p is a tumor suppressor in ECD through the CXCL10-ERK-LIN28a-let7 axis, highlighting another layer of post-transcriptional regulation in this disease. Upregulation of miR-15a-5p in ECD patients may have a potential therapeutic role.

The spillover of animal coronaviruses (aCoVs) to humans has caused SARS, MERS, and COVID-19. Although antibody responses displaying cross-reactivity between SARS-CoV-2 and seasonal/common cold human coronaviruses (hCoVs) have been reported, potential cross-reactivity with aCoVs and the diagnostic implications are incompletely understood. Here, we probed for antibody binding against all 7 hCoVs and 49 aCoVs represented as 12,924 peptides within a phage-displayed antigen library. Antibody repertoires of 269 recovered patients with COVID-19 showed distinct changes compared with 260 unexposed prepandemic controls, not limited to binding of SARS-CoV-2 antigens but including binding to antigens from hCoVs and aCoVs with shared motifs to SARS-CoV-2. We isolated broadly reactive monoclonal antibodies from recovered patients with COVID-19 who bind a shared motif of SARSCoV-2, hCoV-OC43, hCoV-HKU1, and several aCoVs, demonstrating that interspecies cross-reactivity can be mediated by a single immunoglobulin. Using antibody binding data against the entire CoV antigen library allowed accurate discrimination of recovered patients with COVID-19 from unexposed individuals by machine learning. Leaving out SARS-CoV-2 antigens and relying solely on antibody binding to other hCoVs and aCoVs achieved equally accurate detection of SARS-CoV-2 infection. The ability to detect SARS-CoV-2 infection without knowledge of its unique antigens solely from cross-reactive antibody responses against other hCoVs and aCoVs suggests a potential diagnostic strategy for the early stage of future pandemics. Creating regularly updated antigen libraries representing the animal coronavirome can provide the basis for a serological assay already poised to identify infected individuals after a future zoonotic transmission event.