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

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 1^st 2018 and September 31^st 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.