Capsule summary Patients with germline mutations in PIK3CD, immunodeficiency, lymphoproliferation, and autoimmunity show a distinct pattern of abnormal B-cell maturation in the bone marrow.
Introduction: Identifying the causes of adult-onset rheumatic diseases remains a challenge, and limits diagnosis, prognosis, and targeted treatment. We hypothesized that mutations in genes regulating the post-translational modification ubiquitin, previously implicated in two autoinflammatory diseases, may define new rheumatic disorders. Methods: We analyzed peripheral blood exome sequence data from 2,560 individuals with inflammation-related diagnoses for deleterious mutations in >800 ubiquitin-related genes. After discovering three patients with novel UBA1 mutations, we identified additional cases based on clinical similarities through screening multiple independent cohorts. Clinical evaluation of all patients combined with Sanger sequencing, digital droplet PCR, immunoblotting, immunohistochemistry, flow cytometry, and transcriptome/cytokine profiling were performed. CRISPR/Cas9 edited zebrafish provided an in vivo model to assess UBA1 gene function. Results: Twenty-eight adult males were identified with somatic mutations at methionine 41 in UBA1, an X-linked gene, encoding the major E1 enzyme that initiates ubiquitylation. Methionine 41 is highly conserved in UBA1, and these mutations were not observed in exome sequences from over 80,000 healthy controls. Among affected individuals, mutations were found in more than half of hematopoietic stem cells, exclusively in peripheral blood myeloid cells, and not in lymphocytes or fibroblasts. The variant allele fraction of UBA1 p.Met41 mutations in peripheral blood ranged from 20-95%. Patients developed an often fatal, treatment-refractory inflammatory syndrome in late adulthood, with fever, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis and some individuals met clinical criteria for relapsing polychondritis, Sweet syndrome, polyarteritis nodosa, or giant cell arteritis. In addition, the majority of subjects developed myelodysplastic bone marrow with cytopenias, characteristic vacuoles in myeloid and erythroid precursors cells, progressive bone marrow failure and thromboembolic disease, and some fulfilled clinical criteria for myelodysplastic syndrome or plasma cell dyscrasia. Transformation into MDS with excess blasts or acute myeloid leukemia did not occur in any case. Mutations at p.Met41, the initiation start site of the canonical cytoplasmic isoform, caused loss of this protein and expression of a novel, catalytically impaired isoform initiated at p.Met67. Mutant peripheral blood cells exhibited decreased ubiquitylation and activated innate immune pathways. Knockout of the zebrafish cytoplasmic UBA1 isoform homologue, but not the nuclear isoform, caused systemic inflammation. These results identify somatic mutations in UBA1 as the cause, and not the consequence, of this inflammatory disease. Conclusions: We have defined a novel disorder, VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic), which connects seemingly unrelated adult-onset inflammatory and hematologic diseases. Our work also reveals somatic mutations in a hematopoietic stem cell as a cause of adult-onset rheumatic syndromes that overlap with hematologic conditions. Identification of UBA1 mutations as a cause of these diseases has important implications for classification, prognosis, and treatment of patients, and for the role of somatic mutations of hematopoietic origin in multi-organ pathophysiology. Figure Disclosures Young: Novartis: Research Funding.
Background Hodgkin lymphoma (HL) is mainly composed of reactive cells. Lymphocytes, macrophages, eosinophils, mast cells, plasma cells, and other stromal cells constitute these supportive or reactive cells. In-situ glycolytic and oxidative phosphorylation metabolism of reactive cells and cancer cells is unknown in HL. We interrogated HL specimens (N=24) to examine the metabolic compartments in-situ in this disease. Introduction High glycolytic activity is seen in the majority of HL on the basis of high uptake of 2-[18] fluoro-2-deoxy-glucose (FDG) on positron emission tomography (PET). The advent of FDG-PET in HL has revealed that these tumors as a unit are glycolytic and staging and assessment of response to treatment has improved. However, it is unknown if cancer cells and reactive cells share a metabolic phenotype. Purpose To study mitochondrial and glycolytic metabolism in HL cancer and reactive cells. Methods/Materials Two immunohistochemical biomarkers of metabolism were employed on HL tumor sections. Translocase of the Outer Mitochondrial Membrane 20 (TOMM20) protein expression is a biomarker of functional mitochondrial mass and oxidative phosphorylation. Monocarboxylate transporter 4 (MCT4) is a biomarker of glycolysis and lactate export. We selected 24 consecutive cases of classical HL based on morphology and immunohistochemical (IHC) staining for CD30, CD15 and CD45. We recorded FDG-PET standard uptake values (SUV) where available for all tumors studied. Immunohistochemical stains were performed on 5-micron thick, formalin-fixed, paraffin-embedded tissue sections using the horseradish peroxidase method. The primary antibodies used were TOMM20 and MCT4 (Santa Cruz). The immunostaining was graded on a scale of 0 to 2+ according to the intensity and percentage of immunoreactive cancer and reactive cells. Results Reed Sternberg and Hodgkin cells (RS/H) had high TOMM20 expression and low MCT4. In fact, the highest TOMM20 expression out of all cells analyzed was found in the RS/H cells in 20 of the 24 samples. In the other 4 samples, cancer and reactive lymphocytes (RL) had similar expression of TOMM20. The tumor stroma or stromal cells in proximity to cancer cells which includes histiocytes (TS/H) had low TOMM20 expression and high MCT4 expression in all 24 samples analyzed. Higher than baseline FDG-PET uptake is a measure of glycolysis and was found in all tumors where FDG-PET was performed. High MCT4 expression was not found in normal stroma or stroma at a distance from cancer cells. Histiocytes always had low TOMM20 expression but MCT4 expression was high in proximity to cancer cells and low at a distance from cancer cells. Conclusion Glycolysis and lactate export occurs in cancer-associated stroma (TS/H) that is spatially linked to mitochondrial metabolism in cancer cells (RS/H) in HL. This suggests that the most FDG-PET avid cells within HL are the reactive cells and not the cancer cells. Also, normalization of FDG-PET uptake when assessing response to treatment suggests reversal to a metabolically normal stroma. Representative images, 60x: Disclosures: No relevant conflicts of interest to declare.
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