Rapid genomic analysis for the early identification of complement abnormalities in adults with transplant associated thrombotic microangiopathy

Abstract

Abstract Background Transplant-associated thrombotic microangiopathy (TA-TMA) is a rare but often fatal complication of allogeneic hematopoietic stem cell transplantation (Allo-SCT). The prevalence of TA-TMA ranges from 3% to 39% of transplants, due to the heterogeneity of diagnostic criteria employed, and the related case-fatality rate has been reported to be up to 84% (Gavriilaki 2018, Sakeillari 2017). To harmonize diagnostic approaches, a recent consensus (Schoettler 2022) was published where, by adapting the Jodele criteria initially developed for pediatric population (Jodele 2014), TA-TMA was diagnosed when at least four of the following features are present: anemia, thrombocytopenia, hypertension, increased LDH, schistocytes, soluble C5b-9 and proteinuria. With this background, given the small literature in the adult setting using the new criteria (Epperla 2020, Acosta-Medina 2025), we applied the EAABMT criteria to a cohort of adult patients with suspected TA-TMA after allo-SCT, aiming to: 1) confirm the applicability and the role of new criteria at identifying patients with TA-TMA, especially those at higher risk of mortality; 2) verify the activation of complement cascade; 2) assess the frequency of genetic abnormalities of complement system and the possibility of achieving diagnosis in less than 72 hours with rapid genomic analysis employing nanopore sequencing Methods Between January 1st 2018 and September 31st 2021, 195 consecutive adult patients received an Allo-SCT at our at Humanitas Cancer Center. When at least 2 of the features of the EAABMT consensus concomitantly occurred, as recommended by Schoettler, additional workup was pursued. A total of 16 patients were suspected of TA-TMA and blood samples analyzed for ADAMTS13, soluble C5b-9 (sC5b-9) complex and complement genetic studies. ADAMTS13 activity plasma levels were measured with a fluorescence resonance energy transfer assay; sC5b-9 plasma levels and Anti-factor H autoantibodies were determined by ELISA; complement genetic studies were performed by next-generation sequencing (NGS), multiplex ligation–dependent probe amplification and nanopore sequencing. The study was approved by the ethics committee (no. TAM ONC/OSS-02/2025). Results Considering the 16 patients with suspected TA-TMA, one was excluded because of ADAMTS13 < 3%, thus affected by thrombotic thrombocytopenic purpura. Median of post-transplant days for suspecting TA-TMA was 80 days (IQ range 57-95 days). Concomitant post-transplant complications occurred in all patients, including GVHD, either acute or chronic in 11, viral infections in 7 and probable invasive fungal disease in 1. Five patients had <4 EAABMT criteria, ruling out TA-TMA; one of them died because of disease relapse and one for acute GVHD. Ten patients had 4 or more EAABMT criteria and were diagnosed with TA-TMA; at one-year post-transplant 7 died, 1 because of disease relapse and 6 due to post-transplant side effects. The cumulative incidence of 1-year non-relapse mortality was 20% in patients with <4 EAABMT criteria and 60% in patients with (≥4 EAABMT criteria (p=0.07). Consistent with TA-TMA diagnosis (≥4 EAABMT criteria), the 10 patients with TA-TMA had plasma levels of sC5b-9 (median 610 ng/mL, range 210-3313 ng/mL) higher than the 5 patients with <4 EAABMT criteria (257 ng/mL, 152-368 ng/mL; p=0.040). In patients with TA-TMA, plasma levels of sC5b-9 were also higher than those of 20 healthy controls (131 ng/mL, 75-242 ng/mL; p<0.001). None of our patients tested positive for autoantibodies to factor H of the IgG, IgM and IgA classes. By NGS, the genetic variants of uncertain significance in complement-related genes were identified: 7 variants in 5 out of 10 patients with confirmed TA-TMA relative to 1 variant in the group of 5 patients with non-confirmed TA-TMA. CFHR3-CFHR1 deletion, identified by MLPA, was present in 4 of 10 patients with TA-TMA and in 2 of the 5 patients with <4 EAABMT criteria. Using EPI2ME pipeline, nanopore sequencing confirmed the results obtained with targeted NGS in less than 72 hours. Conclusion Our findings support the diagnostic utility of EAABMT-based clinical criteria in adults with suspected TA-TMA and add novel evidence on the possibility to rapidly discover complement genetic abnormalities by rapid genomic analysis with nanopore sequencing. The role of complement genetic abnormalities in TA-TMA pathophysiology is further supported by elevated levels of sC5b-9, marker of complement activation.