MEFV E148Q-P369S-R408Q germ line variant identified from the adolescent imcd-tafro family conferred severe disease manifestations in a cohort of 37 patients with castleman disease Free

Background:Idiopathic multicentric Castleman disease with TAFRO (Thrombocytopenia, Anasarca, Fevers, Reticulin myelofibrosis, Organomegaly) (iMCD-TAFRO) is a group of uncommon lymph-proliferative disorders that could be life-threatening. Adolescent TAFRO is exceptionally rare and effective therapies remain lacking. Pathogenesis of Castleman disease (CD) appears to be largely unknown.

Methods: Clinical data from a retrospective cohort of 37 patients with CD were collected. Blood and/or lymph node biopsy specimens were obtained for whole-exome sequencing (WES). In vitro lipopolysaccharide (LPS) stimulation experiment and single-cell RNA-sequencing (scRNA-seq) were performed to characterize the immune signature using peripheral blood mononuclear cells (PBMC) from an adolescent TAFRO patient (during flare and remission) and his asymptomatic parents, together with a healthy control. The relative gene expression were examined by quantitative PCR. Cytokine levels were assessed using Luminex. This study was approved by the Institutional Review Boards, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, complying with the Declaration of Helsinki.

Results: A previously healthy 15-year-old Chinese male was admitted with chief complaint of abdominal pain and fever. Laboratory tests, imaging examinations, bone marrow and lymph node biopsy confirmed the diagnosis of iMCD-TAFRO. The siltuximab-based anti-IL-6 regimen demonstrated promising clinical efficacy, with the patient maintaining complete remission for 36 months of follow-up at the time of abstract preparation. Using WES, we identified Mediterranean fever (MEFV) E148Q-P369S-R408Q germ line variant in the patient, inherited from his asymptomatic parents. The LPS experiment revealed a more pronounced pro-inflammatory response with remarkable up-regulation of IL-6, IL-1β, and other cytokines from this patient compared to his parents or a healthy control. Colchicine exhibited potency in inhibiting cell aggregation and inflammation activation driven by LPS, suggesting therapeutic potential.

Using scRNA-seq, we found naïve B/memory B cells were increased in the flare of patient compared to his parents and control, showing the highest expression of IL-6. In addition, CD16+ monocytes were characterized by high inflammatory and IL-6 pathway scores, likely contributing to the amplification of the inflammatory response via strong interaction with naïve B/memory B cells. Importantly, MEFV were exclusively enriched in CD16⁺ monocytes, suggesting its role in inflammose signaling. Therapy-induced remission restored the altered condition, corrected the dysregulation of immune cell composition, and alleviated immune malfunction. In addition, we provided the first evidence of megakaryocytes implicated in inflammation via the CXCL signaling network in TAFRO.

Furthermore, in a cohort enrolled with 77 CD patients from Jan 1^st2012 to Dec 31^st 2022, available lymph node biopsy specimens from 37 patients were sequenced by WES. MEFV variants were identified in 76% (28/37) of patients, among which MEFV E148Q-P369S-R408Q accounting for 19% (7/37) of all cases and 25% (7/28) of MEFV mutant patients. Furthermore, we found E148Q-P369S-R408Q was prevailed in 16% (4/25) of UCD, 25% (3/12) of MCD. Strikingly, the variant frequency increased to 50% (2/4) in TAFRO patients. When looking into the 7 patients positive for E148Q-P369S-R408Q, 29% (2/7) were TAFRO subtype.

More important, MEFV E148Q-P369S-R408Q positive CD exhibited a more severe disease course, with higher number of WBC, CRP and lower number of Hb, PLT, Alb and inferior renal function.

Conclusion: Our study adds to the very limited literature by describing the genomic characteristics of an adolescent TAFRO and the high responsiveness to anti-IL-6 based therapy. MEFV E148Q-P369S-R408Q germ line variant was identified and appeared to be potent in promoting inflammation induced by LPS, which was partly abrogated by colchicine in vitro. ScRNA-seq revealed that MEFV-enriched CD16+ monocytes interacted with naïve B/memory B cells, contributing to IL-6 pathway activation. The high incidence of MEFV variants and a more severe disease course with MEFV E148Q-P369S-R408Q in a cohort of 37 CD, suggested a potential role in CD pathogenesis. Thus, this study presents one of the largest cohorts demonstrating the high prevalence of MEFV variants in CD, providing important novel insights for understanding and treating CD, particularly TAFRO.