Citations

Ewing sarcoma is a rare and deadly pediatric bone cancer for which survival rates and treatment options have stagnated for decades. Ewing sarcoma has not benefited from immunotherapy due to poor understanding of how its immune landscape is regulated. We recently reported that ubiquitin-specific protease 6 (USP6) functions as a tumor suppressor in Ewing sarcoma, and identified it as the first cell-intrinsic factor to modulate the Ewing sarcoma immune tumor microenvironment (TME). USP6 induces intratumoral infiltration and activation of multiple innate immune lineages in xenografted nude mice. Here we report that natural killer (NK) cells are essential for its tumor-inhibitory functions, as NK cell depletion reverses USP6-mediated suppression of Ewing sarcoma xenograft growth. USP6 expression in Ewing sarcoma cells directly stimulates NK cell activation and degranulation in vitro, and functions by increasing surface levels of multiple NK cell-activating ligands. USP6 also induces surface upregulation of the receptor for the apoptosis-inducing ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), providing an additional route for enhanced sensitivity to NK cell killing. Furthermore, USP6-expressing Ewing sarcoma and NK cells participate in a paracrine immunostimulatory feedforward loop, wherein IFNγ secreted by activated NK cells feeds back on USP6/Ewing sarcoma cells to induce synergistic expression of chemokines CXCL9 and CXCL10. Remarkably, expression of USP6 in subcutaneous Ewing sarcoma xenografts induces systemic activation and maturation of NK cells, and induces an abscopal response in which growth of distal tumors is inhibited, coincident with increased infiltration and activation of NK cells. This work reveals how USP6 reprograms the Ewing sarcoma TME to enhance antitumor immunity, and may be exploited for future therapeutic benefit. Significance: This study provides novel insights into the immunomodulatory functions of USP6, the only cancer cell–intrinsic factor demonstrated to regulate the immune TME in Ewing sarcoma. We demonstrate that USP6-mediated suppression of Ewing sarcoma tumorigenesis is dependent on NK cells. USP6 directly activates NK cell cytolytic function, inducing both intratumoral and systemic activation of NK cells in an Ewing sarcoma xenograft model.

Abstract Objectives We investigated coronavirus disease 2019 (COVID-19) vaccine safety in pregnant and breastfeeding women with autoimmune diseases (AID) in the COVID-19 Vaccination in Autoimmune Diseases (COVAD) study. Methods Delayed-onset (>7 days) vaccine-related adverse events (AE), disease flares and AID-related treatment modifications were analysed upon diagnosis of AID vs healthy controls (HC) and the pregnancy/breastfeeding status at the time of at least one dose of vaccine. Results Among the 9201 participants to the self-administered online survey, 6787 (73.8%) were women. Forty pregnant and 52 breastfeeding patients with AID were identified, of whom the majority had received at least one dose of COVID-19 vaccine (100% and 96.2%, respectively). AE were reported significantly more frequently in pregnant than in non-pregnant patients (overall AE 45% vs 26%, P = 0.01; minor AE 40% vs 25.9%, P = 0.03; major AE 17.5% vs 4.6%, P < 0.01), but no difference was found in comparison with pregnant HC. No difference was observed between breastfeeding patients and HC with respect to AE. Post-vaccination disease flares were reported by 17.5% of pregnant and 20% of breastfeeding patients, and by 18.3% of age- and disease-matched non-pregnant and non-breastfeeding patients (n = 262). All pregnant/breastfeeding patients who experienced a disease flare were managed with glucocorticoids; 28.6% and 20% of them required initiation or change in immunosuppressants, respectively. Conclusion This study provides reassuring insights into the safety of COVID-19 vaccines administered to women with AID during the gestational and post-partum periods, helping overcome hesitant attitudes, as the benefits for the mother and for the fetus by passive immunization appear to outweigh potential risks.

BACKGROUND: The prognostic role of the congenital heart disease (CHD) anatomic/physiologic classification has not been systematically studied. The purpose of this study was to determine whether CHD physiologic stage provided improvement in prognostic power (to predict all-cause mortality) beyond conventional clinical risk models. METHODS: Retrospective study of adults with CHD at the Mayo Clinic (2003–2019). The CHD physiologic stage was assessed at baseline and 36 (24–48) months, and patients were classified into stages A to D at these time points. Clinical stability (remaining in the same stage), clinical improvement (moving to less advanced stage), and clinical deterioration (moving to more advanced stage) were determined at 36 months. We defined conventional clinical risk indices as age/sex, functional class, comorbidities, cardiac procedures, hepatorenal dysfunction, and ventricular/valvular dysfunction. RESULTS: Of 5321 patients, 1649 (31%), 1968 (37%), 1224 (23%), and 480 (9%) were in stages A, B, C, and D at baseline. Of 5321 patients, 4588 (86%) also had assessments at 36 months, and of these patients, 3347 (73%), 386 (8%), and 855 (19%) had clinical stability, deterioration, and improvement, respectively. Patients with clinical improvement were more likely to have undergone cardiac procedures between both assessments. Both baseline CHD physiologic stage (hazard ratio, 1.13 [95% CI, 1.09–1.17]; P <0.001, per unit increase in stage) and change in CHD physiologic stage (hazard ratio, 1.46 [95% CI, 1.32–1.61]; P =0.007, per unit increase in stage) were associated with mortality after adjustments for conventional risk indices and provided incremental improvement in prognostic power beyond conventional clinical risk models as evidence by an increase in C statistic from 0.702 (0.681–0.724) to 0.769 (0.754–0.787). CONCLUSIONS: The CHD physiologic stage can potentially be used for risk stratification, as well as to monitor disease progression and response to therapy.

AbstractContextInhibition of the neonatal fragment crystallizable receptor (FcRn) reduces pathogenic thyrotropin receptor antibodies (TSH-R-Ab) that drive pathology in thyroid eye disease (TED).ObjectiveWe report the first clinical studies of an FcRn inhibitor, batoclimab, in TED.DesignProof-of-concept (POC) and randomized, double-blind placebo-controlled trials.SettingMulticenter.ParticipantsPatients with moderate-to-severe, active TED.InterventionIn the POC trial, patients received weekly subcutaneous injections of batoclimab 680 mg for 2 weeks, followed by 340 mg for 4 weeks. In the double-blind trial, patients were randomized 2:2:1:2 to weekly batoclimab (680 mg, 340 mg, 255 mg) or placebo for 12 weeks.Main OutcomeChange from baseline in serum anti-TSH-R-Ab and total IgG (POC); 12-week proptosis response (randomized trial).ResultsThe randomized trial was terminated because of an unanticipated increase in serum cholesterol; therefore, data from 65 of the planned 77 patients were analyzed. Both trials showed marked decreases in pathogenic anti-TSH-R-Ab and total IgG serum levels (P &lt; .001) with batoclimab. In the randomized trial, there was no statistically significant difference with batoclimab vs placebo in proptosis response at 12 weeks, although significant differences were observed at several earlier timepoints. In addition, orbital muscle volume decreased (P &lt; .03) at 12 weeks, whereas quality of life (appearance subscale) improved (P &lt; .03) at 19 weeks in the 680-mg group. Batoclimab was generally well tolerated, with albumin reductions and increases in lipids that reversed upon discontinuation.ConclusionsThese results provide insight into the efficacy and safety of batoclimab and support its further investigation as a potential therapy for TED.

AbstractBackground and PurposeTo determine the incidence of acute neuroimaging (NI) findings and comorbidities in the coronavirus disease of 2019 (COVID‐19)‐infected subjects in seven U.S. and four European hospitals.MethodsThis is a retrospective study of COVID‐19‐positive subjects with the following inclusion criteria: age >18, lab‐confirmed COVID‐19 infection, and acute NI findings (NI+) attributed to COVID‐19 on CT or MRI brain. NI+ and comorbidities in total hospitalized COVID‐19‐positive (TN) subjects were assessed.ResultsA total of 37,950 COVID‐19‐positive subjects were reviewed and 4342 underwent NI. NI+ incidence in subjects with NI was 10.1% (442/4342) including 7.9% (294/3701) in the United States and 22.8% (148/647) in Europe. NI+ incidence in TN was 1.16% (442/37,950). In NI (4342), incidence of ischemic stroke was 6.4% followed by intracranial hemorrhage (ICH) (3.8%), encephalitis (0.5%), sinus venous thrombosis (0.2%), and acute disseminated encephalomyelitis (ADEM) (0.2%). White matter involvement was seen in 57% of NI+. Hypertension was the most common comorbidity (54%) before cardiac disease (28.8%) and diabetes mellitus (27.7%). Cardiac disease (p < .025), diabetes (p < .014), and chronic kidney disease (p < .012) were more common in the United States.ConclusionThis multicenter, multinational study investigated the incidence and spectrum of NI+ in 37,950 hospitalized adult COVID‐19 subjects including regional differences in incidences of NI+, associated comorbidities, and other demographics. NI+ incidence in TN was 1.16% including 0.95% in the United States and 2.09% in Europe. ICH, encephalitis, and ADEM were common in Europe, while ischemic strokes were more common in the United States. In this cohort, incidence and distribution of NI+ helped characterize the neurological complications of COVID‐19.

PURPOSE Erb-B2 receptor tyrosine kinase 2 ( ERBB2)–positive breast cancer (BC) is particularly common in young women. Genomic features of ERBB2-positive tumors before and after chemotherapy and trastuzumab (chemo + H) have not been described in young women and are important for guiding study of therapeutic resistance in this population. METHODS From a large prospective cohort of women age 40 years or younger with BC, we identified patients with ERBB2-positive BC and tumor tissue available before and after chemo + H. Whole-exome sequencing (WES) was performed on each tumor and on germline DNA from blood. Tumor-normal pairs were analyzed for mutations and copy number (CN) changes. RESULTS Twenty-two women had successful WES on samples from at least one time point; 12 of these had paired sequencing results from before and after chemo + H and 10 had successful sequencing from either time point. TP53 was the only significantly recurrently mutated gene in both pre- and post-treatment samples. MYC gene amplification was observed in four post-treatment tumors. Seven of 12 patients with paired samples showed acquired and/or clonally enriched alterations in cancer-related genes. One patient had an increased clonality putative activating mutation in ERBB2. Another patient acquired a clonal hotspot mutation in TP53. Other genomic changes acquired in post-treatment specimens included alterations in NOTCH2, STIL, PIK3CA, and GATA3. There was no significant change in median ERBB2 CN (20.3 v 22.6; Wilcoxon P = .79) between paired samples. CONCLUSION ERBB2-positive BCs in young women displayed substantial genomic evolution after treatment with chemo + H. Approximately half of patients with paired samples demonstrated acquired and/or clonally enriched genomic changes in cancer genes. ERBB2 CN changes were uncommon. We identified several genes warranting exploration as potential mechanisms of resistance to therapy in this population.

Purpose: To describe the characteristics and outcomes of eyes with idiopathic full-thickness macular holes that underwent initial medical management. Methods: This retrospective study included eyes with full-thickness macular holes that were initially managed with 1 month of topical therapy. Eligible subjects were treated with dorzolamide 2% three times a day, nepafenac 0.1% twice a day, and prednisolone acetate 1% four times a day. The primary end points was hole closure at one month and secondary end point was change in best-corrected visual acuity. Results: Ten subjects (mean age: 62.80 years; female: 50%) with unilateral full-thickness macular holes were studied. The mean basal diameter of the entire cohort at baseline was 824.1 µm (median 828 µm). Four (40%) of the smaller holes (mean 698 µm; median 698.50 µm) closed after 1 month of topical therapy, whereas larger holes (mean 908.17 µm; median 889.50 µm) did not close. In one eye, the hole reopened 4 months after stopping the medication but closed again at 1 month after restarting the topical treatment. Median best-corrected visual acuity improved from 0.35 logMAR at baseline to 0.05 logMAR in eyes that closed but remained at 0.70 logMAR at 1 month in eyes that did not close. Conclusion: Topical corticosteroid, nonsteroidal antiinflammatory, and carbonic anhydrase inhibitor therapy may promote closure of small full-thickness macular holes, but large holes are less likely to respond. One month of topical therapy might avoid subjecting some patients to complex vitreoretinal surgery without compromising visual outcomes. Macular hole may reopen after stopping the topical therapy.

Abstract An important aspect to determine the efficacy of a targeted macromolecule in treating brain tumors is penetration and activity in tumor across a heterogeneously intact blood-brain barrier. In this study, novel epidermal growth factor receptor (EGFR)-targeted antibody drug conjugates (ADCs) were constructed with a focus on cytotoxicity in glioblastoma (GBM) with an EGFR heterogeneous cell population. Initially, the cytotoxicity of free payloads with multiple mechanisms of cell kill was assessed. These included tesirine (TS, induces DNA cross-links), deruxtecan (Dxd) and SN38 (inhibit topoisomerase I), and an auristatin (MMAE) and maytansinoid (DM1) (inhibit microtubule polymerization). Across four GBM PDXs with varied EGFR expression (GBM6, 39 and 108- EGFRviii amplified; GBM10 - non-amplified), free TS and MMAE were consistently potent with EC50 values ranging from 0.50-45.9 pM and 7.9-229.1 pM, respectively. In contrast, the other free toxins were less potent; EC50 values: Dxd, 0.21-13.1 nM; SN38, 0.87-5.6 nM; DM1, 2.1-19.9 nM. Based on these results, the 40H3 EGFR-specific IgG was used to construct ADCs with TS and MMAE. 40H3-TS had a drug:antibody ratio (DAR) of 2.5 and was potently cytotoxic in GBM6, GBM39 and GBM108, while minimal cytotoxicity was observed in GBM10 or normal astrocyte SVG-A cells. 40H3-MMAE had a DAR of 3 and was similarly effective in GBM6 and GBM39 but less potent in GBM108, GBM10, and SVG-A. Bystander cytotoxicity was evaluated U87 cells expressing eGFP/fLuc2 (U87eGFP/fluc2) or EGFRviii (U87EGFRviii). Using live-cell imaging, U87EGFRviii cells treated with 40H3-TS or 40H3-MMAE had significantly reduced cell confluence relative to control. The same drug treatments in U87eGFP/fLuc2 cells had no effect on growth/confluence. However, in a 1:1 mixed culture, overall confluence was reduced from 89% in control to 35% and 32% after 40H3-TS (p&lt;0.0001) or 40H3-MMAE (p&lt;0.0001) treatment, respectively. This bystander killing of U87eGFP/fLuc2 cells was indicated by a reduction in confluence of green-fluorescent cells from 50% in control versus 29% with 40H3-TS (p&lt;0.0001) and 26% with 40H3-MMAE (p&lt;0.0001) treatment. In GBM39 orthotopic tumors, a single infusion of 10 or 20 µg 40H3-TS via convection enhanced delivery (CED) reduced the bioluminescence signal 7 days post treatment by ~10-fold (p=0.03) as compared to 40H3 control. However, 50% and 80% mortality was observed within a week of infusing 10 and 20 µg 40H3-TS, respectively. Neurotoxicity was associated with neuron loss in treated hemisphere as determined by NeuN staining. In summary, these data highlight the potential for novel EGFR-targeted ADCs to provide potent direct and bystander cytotoxicity to GBM cells. However, further selection and optimization of the conjugated toxins will be required to balance potency and bystander killing with toxicity for EGFR-targeted ADCs. Citation Format: Sonia Jain, Eric Chun Ho, Kendra A. Porath, Antonella Antignani, David J. FitzGerald, Jann N. Sarkaria. Bystander effects of EGFR-targeting 40H3 antibody-drug conjugates in glioblastoma with heterogeneous EGFR expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 556.